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.

External ID: HMS1262

Protocol: NEC is stored at -80 degrees at a concentration of 15mg/ml in single use aliquots.

On the day of the screen, 20ul of purified NEC is aliquoted using a Multidrop Combi reagent dispenser into 384 well plates (Corning 3824). 100nl of compound dissolved in DMSO was transferred to each well of the assay plated via pin transfer. The plates (NEC + compound) are incubated at room temperature for 3 hours. Acceptor and donor reagents (CisBio 620/665 pair) are combined then added to each well at 5 microL volumes at a concentration of 8 nM and 80nM respectively. The plates are spun at 1k rpm for 1 min and incubated overnight at 4 degrees, then for one hour the subsequent day at room temperature.

Flourescent measurements are read on the Envision 1 plate reader at ICCB-L. The raw data consists of two fluorescence readings - at 665 nm and 620 nm for the acceptor and donor respectively.

Comment: Data analysis:
The raw data consists of two fluorescence readings - at 665 and 620 nm for the acceptor and donor respectively. The data is processed as a ratio of the emission from the acceptor over the donor (homogeneous time resolved fluorescence ratio). Normalized percent inhibition (NPI) for all experimental wells is calculated based on plate averages for negative and positive control HTRF ratio. Positives are scored as any ratio with a 50% or greater inhibition as compared with the positive control (i.e. NEC + Untagged UL50). To be considered a hit, both replicates need to score as positive. Activity scores are derived from NPI, with 100 = 100% inhibition (> 100% set to 100) and 0 = no inhibition (< 0% set to 0). Note that some compounds with NPI <50% (activity scores < 50) are classified as potential hits based on additional criteria (typically by selecting wells with low ratios compared to other experimental wells on the plate).

External ID: epac2-activator-v2

Protocol: Briefly, three uL of reagents (100 nM EPAC2, 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: epac2-inhibitor-v2

Protocol: Briefly, three uL of reagents (100 nM EPAC2, 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 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: 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: 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: BCAT SD DP Set

Protocol: Cell Culture: HCT116 cells lacking activated Beta Catenin were subcultured every 3-4 days in McCoy's 5A medium with 10% fetal bovine serum, 100 U/mL penicillin, 100 ug/mL streptomycin and 2 mM glutamine (complete growth medium), incubated at 37 degrees C in 5% carbon dioxide, and maintained at sub-confluent density for no more than 20 passages.

Compound Dosing/Plating: Carrier or sulindac sulfide (Sigma-Aldrich) were diluted in complete growth medium to prepare a 3X concentrated dosing solution (300 uM sulindac sulfide /0.3% DMSO) which was dispensed into 96-well black clear-bottom tissue culture treated plates (50 uL volume). This resulted in a final culture volume in assay plates of 150 uL, with final concentrations of 100 uM sulindac sulfide (0.1% DMSO) for the controls and 10 uM (0.1% DMSO) for the library compounds.

Cell Plating: One hundred uL of complete growth medium containing 5,000 cells were dispensed per well, while stirring. Plates were incubated at room temperature for 30 min prior to incubation at 37 degrees C in 5% carbon dioxide for 60 h.

Endpoint/Detection: At the end of the treatment period, assay plates were removed from the incubator and equilibrated to room temperature 10 min. One hundred uL of Cell Titer Glo reagent was added and plates were incubated for an additional 10 min in the dark. At the end of the incubation, assay plates were analyzed using a PerkinElmer Victor3V microplate reader with an integration time of 1.0 s in luminescence mode.

Comment: Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.

Outcome:
The effect of library compounds at 10 uM on viability of HCT116 cells expressing activated Beta Catenin was calculated as follows:

% Viability = 100*(Cmpd Lum) /(Median Cell Ctrl Lum )
Compounds that reduced viability to <= 29.74% were scored as active (the median viability value for all experimental compounds [96.32%] minus 3 times the standard deviation [22.19%]).

Based on a 3-tiered scoring system, primary screening data received a score from 0 to 40 as an inverse function of % viability, where 100% viability or higher corresponds to a score of 0 and 0% viability corresponds to a score of 40.

External ID: HMS1485

Protocol: Prior to screening, HeLa cells were transduced with a lentivirus carrying the vector PHAGE-N CMVt N-RIG3 p53(R273C) encoding constitutively expressed SGFP2 fused to histone 2B and mRuby-p53(R273C). Following transduction, the cells were selected using neomycin and fluorescent activated cell sorting. The day before the compound treatment, cells were plated (750 cells/well) in a final volume of 30 L/well of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum using a Thermo Combi. Plates were spun for 45 s at 500 rpm and incubated overnight (~24 h) at 37 degrees C, 5% CO2.

On the day of the screening, 33 nL of each compound was transferred to assay wells via a custom pin-transfer workstation. Additionally, 33 nL of positive (Velcade 300 M) and negative (DMSO 100%) controls were transferred to each plate into the corresponding columns that did not contain experimental compounds. For every compound library plate, there were two daughter plates (A and B). Plates were stacked 5 high, covered with lids, and incubated at 37 degrees C for 24h and 48h.

Assay plates were read using an Acumen Laser Scanning Cytometer 24h and 48h after compound treatment, using the following settings: For SGFP2 (channel 2), the 488 nm laser was used, with 6 mW output, 600 voltage gain and sensitivity threshold at 1; and for mRuby (channel 3), the 561 nm laser was used, with 8 mW output, 600 voltage gain and sensitivity threshold at 1.

Comment: Total nuclei and %p53 positive was calculated for all wells at 24 and 48h using the Cellista software. Percentage of p53 positive cells values were normalized to the positive and negative controls to determine a normalized percent of p53 activation. This was done on a per plate basis. The proteasome inhibitor Velcade (Adipogen, AG-CR1-3602-M005) at 330 nM was used as positive control while only the vehicle (DMSO, Sigma, D8418, 0.1% final concentration) was used as negative control. From these values the following normalized values were calculated (for both 24h and 48h): Normalized total nuclei = (total nuclei in experimental well / plate average negative control total nuclei) * 100. Normalized %p53 positive = (%p53 positive in experimental well - plate average negative control %p53 positive cells) / (plate average positive control %p53 positive cells - plate average negative control %p53 positive cells) * 100.

Based on the normalized percent p53 positive values, a compound was considered active when replicate average normalized % p53 positive cells > 50% for either or both time points. Activity scores were based on the replicate average normalized %p53 positive cells at 24hr. Values less than 0 were set to 0, and values > 100 were set to 100 (100% activity). Compounds with an activity score > 50 were considered active; some compounds were scored active despite having an activity score < 50 since % p53 positive cells was > 50% only at 48hr.

The average normalized total nuclei values were categorized as:
Very Low: x < 25%
Low: 25% < X > 50%
Medium: 50% < X > 75%
High: x < 75%

The average normalized % of p53 positive cells values were categorized as:
Low: x < 25%
Medium: 25% < X > 50%
Strong: 50% < X > 75%
Very Strong: x < 75%

Compounds that scored as strong or very strong in this classification (at either time point) were selected for follow up.