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: TELO_02

Protocol: HCT116 colorectal cancer cells are infected with Ad-hTR-mut and cytotoxicity is assessed 5-days post infection, measured by standard MTT assay. Ad-hTR-mut constructed using the Adeasy system (Agilent) and purified and quantified using Adeno-x purification and rapid titre kits (Clontech). Cells are seeded at 10000 cells per well and infected with 500pfu/cell in 96 well plates (Costar) then incubated for 48h prior to addition of compounds. Compounds were screened at 10uM in duplicate 2 days post-infection and MTT assay performed 3 days later at 5 days post-infection. A negative control adenovirus was included on every plate as a control for non-specific viral toxicity and all wells were expressed relative to untreated cells. Absorbance at 570nm was determined using a Molecular Devices spectramax plus instrument and analysed using SoftMax Pro software. Hits were taken to be compounds giving greater than 2.5-fold increase in absorbance indicating increased cell survival.

Comment: Target cell viability at time of assay was 30-50% after infection with this stock of Ad-hTR-mut. Compounds giving 2.5-fold increase in survival relative to this condition were taken to be hits.

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: HMS1158_MLP

Protocol: A 5 mL overnight pre-culture of E. coli is started by inoculating a tube of lysogeny broth with a single colony from an agar plate. The tube is incubated at 37 degrees C overnight with shaking. The resulting saturated culture is removed from the shaker and diluted into fresh lysogeny broth at 1:100 proportion and incubated at 37 degrees C with shaking for two hours. The culture is removed from the shaker and absorbance is measured at 600 nm. The culture is diluted into fresh lysogeny broth to an estimated absorbance of 0.0002. For each library plate to be screened, two 384-well assay plates are filled with 30 microL per well of fresh lysogeny broth, except for the positive control columns, which are left empty. The multiple of two is necessary to expose the strain in duplicate to the same compounds from a given library plate.

300 nL of experimental compounds are transferred into the assay plates from library plates using a robotically manipulated stainless steel pin array, exposing the strain to the same compounds at the same concentration. The positive control columns are filled with 30 microL of the positive control compound in LB. Thirty microL of E. coli culture at absorbance of 0.0002 are then added to the assay plates, producing a total volume of 60 microL in each well. Plates are incubated at 37 degrees C without shaking and with humidification for 23 hours. The plates are removed from the incubator, and absorbance of the cultures is measured at 600 nm using a PerkinElmer EnVision plate reader. The absorbance is used as the readout.

Positive control: (R)-4-(4-(2-fluoro-4-methoxyphenyl)-2-oxopyridin-1(2H)-yl)-N-hydroxy-2-methyl-2-(methylsulfonyl)butanamide was present at 0.0754 microM in all wells of column 24.

Negative control: No treatment in remaining empty wells of the assay plate (minimally, wells in column 23).

Comment: A threshold constant was calculated by subtracting 3.6 X experimental well standard deviation for absorbance from the experimental well average absorbance, combining all plates in a screening run. Well absorbance was then subtracted from this threshold value. Wells were considered active if the resulting value after subtracting the threshold constant > 0 for at least one replicate. Activity scores were calculated by subtracting well absorbance from negative control plate average absorbance, dividing by the difference between plate negative and positive control plate average absorbance, and multiplying by 100. Resulting values > 100 were set to 100 and < 0 were set to 0, where 100 = 100% activity.

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: 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.