External ID: TRXR101

Protocol: Assay protocol: 2 uL of reagents (buffer in column 4 as negative control and 90 nM rTrxR1 in columns 1-3 and 5-48) were dispensed into Greiner black solid-bottom 1,536-well assay plates, followed by 1 uL of NADPH (400 uM final concentration) to each well. The plates were centrifuged at 1000 rpm for 15 seconds and subsequently incubated for 5 min at room temperature (~22 deg C) to allow for rTrxR1 reduction. Compounds (23 nL) were then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). In addition, a duplicate 2-fold serial dilution of the control compounds auranofin, a known gold-based TrxR1 inhibitor, and juglone (5-hydroxy-1,4-naphthoquinone), a natural TrxR1 substrate, were pin-transferred to columns 2 and 3, respectively. After incubation for 15 min at room temperature (~22 deg C), 1 uL of selenite (400 uM final concentration) were dispensed to each well. The plate was immediately transferred to a ViewLux high-throughput CCD imager (PerkinElmer), wherein kinetic measurements of NADPH fluorescence (Ex 340 nm, Em 450 nm) were acquired (8 minute kinetic read, see Table 1). Read 1 was utilized to assess the capacity of a compound to serve as an rTrxR1 substrate, i.e. a decrease in NADPH fluorescence compared to the no-compound background is an indication of a substrate behavior for that particular compound. For inhibitory activity of a compound, delta values, computed as the difference in fluorescence intensity between the first and last reads of an 8-minute time kinetic window, were used. All reagents were diluted in an assay buffer consisting of 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 0.01% Tween-20.

Throughout the screen, reagent bottle and all liquid lines were made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a 'double-pinning' step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the columns 5-48, were inserted uniformly at the beginning and the end of each library in order to monitor and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity over time. Screening data were corrected, normalized, and concentration-effect relationships were derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts).

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: 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: TDP1640

Protocol: Buffer: 1X PBS, pH 7.4, 80 mM KCl, 0.05% Tween-20

Reagents/Controls:
[1] 3 ul of buffer is dispensed in columns 3 & 4 as negative control (no enzyme and maximum signal),
[2] 3 ul of enzyme (1 nM final) is dispensed in columns 1,2, 5-48,
[3] 1 ul of substrate (15 nM final) is dispensed throughout the plate,
[4] 1 ul of AS donor/acceptor bead mix (10 ug/ml final) is dispensed throughout the plate

Assay steps:
3 ul of Tdp1 enzyme is dispensed to 1536-well Kalypsys black solid bottom plates. Compounds and controls (23 nl) are transferred via Kalypsys Pin Tool. The plates are incubated for 15 min at room temperature, and then 1 ul of substrate is added to start the reaction. After 5 min incubation at room temperature, 1 ul of AS donor/acceptor bead mix is added and the plates are further incubated for 10 min at room temperature. The detection is then performed on a PerkinElmer Envision 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: 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: Vaccinia-p2mCherry

Protocol: A549 cells were plated at a concentration of approximately 6,000 cells per well in clear-bottom black-walled plates. Compounds were added to the assay plate followed by addition of virus (LREV) that expresses the Venus fluorescent protein early in viral ininfection. During the late viral infection, mCherry was also added to the plate at an MOI of 10 (~ 60,000 plaque forming units (PFU) of virus). Following virus addition, plates were incubated in a tissue-culture incubator for 12-18 hours. At increasing times postinfection, fluorescence from both Venus and mCherrry was determined using a plate-reader and standard conditions for observing Venus fluorescence (EX515/EM530) and mCherry fluorescence (EX587/EM610). In each plate, positive controls (virus infection in the presence of 1% DMSO only) and an inhibitory drug control (AraC at 500 nM) was included and used to normalize the data.

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: 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.

External ID: TRXR100

Protocol: Assay protocol: 2 uL of reagents (buffer in column 4 as negative control and 90 nM rTrxR1 in columns 1-3 and 5-48) were dispensed into Greiner black solid-bottom 1,536-well assay plates, followed by 1 uL of NADPH (400 uM final concentration) to each well. The plates were centrifuged at 1000 rpm for 15 seconds and subsequently incubated for 5 min at room temperature (~22 deg C) to allow for rTrxR1 reduction. Compounds (23 nL) were then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). In addition, a duplicate 2-fold serial dilution of the control compounds auranofin, a known gold-based TrxR1 inhibitor, and juglone (5-hydroxy-1,4-naphthoquinone), a natural TrxR1 substrate, were pin-transferred to columns 2 and 3, respectively. After incubation for 15 min at room temperature (~22 deg C), 1 uL of selenite (400 uM final concentration) were dispensed to each well. The plate was immediately transferred to a ViewLux high-throughput CCD imager (PerkinElmer), wherein kinetic measurements of NADPH fluorescence (Ex 340 nm, Em 450 nm) were acquired (8 minute kinetic read, see Table 1). Read 1 was utilized to assess the capacity of a compound to serve as an rTrxR1 substrate, i.e. a decrease in NADPH fluorescence compared to the no-compound background is an indication of a substrate behavior for that particular compound. For inhibitory activity of a compound, delta values, computed as the difference in fluorescence intensity between the first and last reads of an 8-minute time kinetic window, were used. All reagents were diluted in an assay buffer consisting of 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 0.01% Tween-20.

Throughout the screen, reagent bottle and all liquid lines were made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a 'double-pinning' step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the columns 5-48, were inserted uniformly at the beginning and the end of each library in order to monitor and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity over time. Screening data were corrected, normalized, and concentration-effect relationships were derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts).

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: Vaccinia-p2Venus

Protocol: A549 cells were plated at a concentration of approximately 6,000 cells per well in clear-bottom black-walled plates. Compounds were added to the assay plate followed by addition of virus (LREV) that expresses the Venus fluorescent protein early in viral infection. During the late viral infection, mCherry was also added to the plate at an MOI of 10 (~ 60,000 plaque forming units (PFU) of virus). Following virus addition, plates were incubated in a tissue-culture incubator for 12-18 hours. At increasing times post-infection, fluorescence from both Venus and mCherrry was determined using a plate-reader and standard conditions for observing Venus fluorescence (EX515/EM530) and mCherry fluorescence (EX587/EM610). In each plate, positive controls (virus infection in the presence of 1% DMSO only) and an inhibitory drug control (AraC at 500 nM) was included and used to normalize the data.

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.