External ID: ADAM17_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that inhibit ADAM17. This assay employs a fluorophore and quencher pair. F =EDANS fluorophore, Q = DABCYL quencher. When intact, EDANS emission at 460nm is quenched by DABCYL via fluorescence resonance energy transfer. Upon cleavage of the scissile bond (A~V) by ADAM protease, the distance between fluorophore and quencher increases resulting in fluorescence increase at 460nm. Compounds are tested in singlicate at a final nominal concentration of 6.95 micromolar.

Protocol Summary:

Prior to the start of the assay, 2.5 microliters 2X ADAM17 enzyme (20 nM in Assay Buffer: 50 mM HEPES, 0.01% Brij, pH 7.5) are dispensed into 1536 microtiter plates. Compounds are added to plate (final concentration TBD) and incubated for 30 minutes at 25 degrees Celsius. The assay is started by dispensing 2.5 microliter of2X DM2 substrate (20 uM in Assay Buffer) to all wells. Plates are centrifuged and after 3 hours of incubation at 25 degrees Celsius, fluorescence is measured (excitation = 359nm, emission = 460nm).

The % inhibition for each well was then calculated as follows:

%_Inhibition = ( RFU_Test_Compound - MedianRFU_Low_Control ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells treated with 1 micromolar Marimastat
Low_Control is defined as wells containing DMSO.


Interval Cutoff:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-8, for inactive 8-0.

List of Reagents:

ADAM17 enzyme (R&D Systems, part # 930-ADB)
EDANS-DABCYL DM2 peptide substrate (Supplied by Assay Provider)
0.5M HEPES solution, pH7.5 (Teknova, part #101319-900)
Brij-35 (Sigma-Aldrich, part # P1254)
1536 well plate (Corning, part # 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

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: EPAC1-agonist-p1

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: 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 activators are ranked higher than compounds that showed apparent inhibition.
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: PLCB3_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of the activity of phospholipase C isozymes, PLC-B3. In this assay, PLC-B3 isozyme is incubated with test compounds and fluorogenic reporter WH-15. As designed, test compounds that act as PLC-B3 inhibitors will prevent the hydrolysis of WH-15 fluorogenic reporter, thus preventing the release of IP3, a quinomethide derivative, and 6-aminoquinoline, which is highly fluorescent, leading to decreasing well fluorescence. Compounds are tested in singlicate at a nominal test concentration of 12.2 micromolar.

Protocol Summary:

Prior to the start of the assay, 2 microliters of PLC-B3 at a final concentration of 0.4ng/ul (in 50 mM HEPES pH 7.2, 70 mM KCl, 3mM CaCL2, 3mM EGTA, 2mM DTT, 0.04mg/mL acid-free BSA, with Cholate 0.5%) are dispensed into 1536 microtiter plates, 1 microliter of assay buffer is dispensed into columns 4-48 and 1 microliter of 0.2M EGTA is added to columns 1-3. Compounds are added to plate (final concentration 12.2uM) and incubated for 10 minutes at 25 degrees Celsius. The assay start by the addition of 2 microliter of WH-15 fluorogenic reporter at a final concentration 10uM in Assay Buffer to all wells. Plates were centrifuged and after 90 min of incubation at 25 degrees Celsius fluorescence is measured at 355nm excitation and 535nm emmision.

The percent inhibition for each compound was calculated as follows:

100 *( ( Test_Compound - Median_Low_Control) / ( Median_High_Control - Median_Low_Control) )

Where:

Test_Compound is defined as wells containing PLCB3 in the presence of test compound and WH15 fluoreogenic reporter.
High_Control is defined as wells containing PLCB3, WH15 fluoreogenic reporter and EGTA.
Low_Control is defined as the median of the wells containing test compounds.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-13, for inactive 13-0.

List of Reagents:

PLCB3 isozyme (Supplied by Assay Provider)
WH-15 fluorogenic reporter (Supplied by KXTBio)
HEPES (Fisher, BP310)
Sodium cholate hydrate (Sigma, C6445)
CaCl2 (Sigma, 06991)
EGTA (Fisher, O2783)
DTT (Fisher, BP172)
KCl (Sigma, P9541)
1536-well plates (Corning, part 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: ADAM10_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that inhibit ADAM10. This assay employs a fluorophore and quencher pair. F =EDANS fluorophore, Q = DABCYL quencher. When intact, EDANS emission at 460nm is quenched by DABCYL via fluorescence resonance energy transfer. Upon cleavage of the scissile bond (A~V) by ADAM protease, the distance between fluorophore and quencher increases resulting in fluorescence increase at 460nm. Compounds are tested in singlicate at a nominal final nominal concentration of 6.95 micromolar.

Protocol Summary:

Prior to the start of the assay, 2.5 microliters 2X ADAM10 enzyme (20 nM in Assay Buffer: 50 mM HEPES, 0.01% Brij, pH 7.5) are dispensed into 1536 microtiter plates. Compounds are added to plate (final concentration TBD) and incubated for 30 minutes at 25 degrees Celsius. The assay is started by dispensing 2.5 microliter of2X DM2 substrate (20 uM in Assay Buffer) to all wells. Plates are centrifuged and after 3 hours of incubation at 25 degrees Celsius, fluorescence is measured (excitation = 359nm, emission = 460nm).

The % inhibition for each well was then calculated as follows:

%_Inhibition = ( RFU_Test_Compound - MedianRFU_Low_Control ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells treated with 1 micromolar Marimastat
Low_Control is defined as wells containing DMSO.

Interval Cutoff:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-19, for inactive 19-0.

List of Reagents:

ADAM10 enzyme (R&D Systems, part # 936-AD)
EDANS-DABCYL DM2 peptide substrate (Supplied by Assay Provider)
0.5M HEPES solution, pH7.5 (Teknova, part #101319-900)
Brij-35 (Sigma-Aldrich, part # P1254)
1536 well plate (Corning, part # 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: Bursicon-induced LGR2 mediated cAMP production in LGR-2/CRE6x-Luciferase co-transfected HEK293 cells Inhibition - 7011-01_Antagonist_SinglePoint_HTS_Activity

Protocol: Protocol

Day 0:
Cell Culture
HEK 293 cells are plated in DMEM culture media (containing 10% Fetal Bovine Serum (FBS) and 1X penn/strep/glutamine) at a density of 5x106 cells/T175 flask and grown for 3 days at 37 degrees C in 5% CO2 incubator.

Day 3:
Transient Transfection
Cells are transiently transfected in DMEM transfection media (containing 1X penn/strep/glutamine without serum) using polyethyleneimine (PEI).
1)Mix 2.9ug/flask of cDNA encoding the wild-type Bursicon Receptor with 14.6ng/flask of cDNA encoding a CRE-luciferase reporter gene with a PEST/HCL sequence in 2mL of transfection media
2)Add 61uL/flask of PEI of 1mg/mL in 1.5mL of transfection media
3)Mix the 2mL cDNA solution with 1.5mL of PEI solution and incubate at RT for 20 mins
4)Aspirated the culture media from the cells in T175 flask
5)Add 25 mL of transfection media and 3.5mL of transfection mixture to T175 falsk.
6)Mix the media with transfection mixture well and transfect for 2 days at 37 degrees C in 5% CO2 incubator

Day 5:
Cell Plating
1)Typsinize the Cells with 5mL of 0.05% Trypsin and incubate at 37oC for 5 min.
2)Add 5mL of DMEM assay media (containing DMEM without phenol red, 10% NuSerum and 1x Pen/Strep/Glutamine) to inactive trypsin
3)Collect cells by centrifugation at 1000rpm for 5 min
4)Suspend the cells in DMEM assay media to a cell density of 4x105 cells/mL
5)Plate the cells to 1536-well Aurora Mako plate with 2000 cells/well/5uL using ViaFill
6)Incubate the cells at 37 degrees C in 5% CO2 incubator on GS system for overnight

Day 6:
Compound Treatment, Agonist Stimulation and Detection
Assay Setup
1)Thaw aliquoted Bursicon conditioned media and diluted with DMEM assay media at a 1/20 dilution. Filter the solution through a 0.22uM filter
2)Add DMEM condition media, the diluted Bursicon conditioned media and SteadyGlo to the bottles and setup BioRAPTR

Run automation protocol on GS system
1)Take the assay plate with transfected cells from the incubator
2)Transfer 5nL/well of 10mM compound to assay plate
3)Incubate 30 mins in incubator
4)Add 1uL/well of DMEM assay media to PosCon wells and diluted Bursicon solution to the other wells using BioRAPTR (Beckman) based on the plate map
5)Incubate the assay plate for 4 h in incubator
6)Take the plate out from the incubator and equilibrate the assay plate at RT for 10 mins
7)Add 1uL/well of SteadyGlo to assay plate using BioRAPTR, incubate at RT for 10 mins
8)Read the plate on ViewLux (PerkinElmer) for Luminescence.

Culture Media
DMEM high glucose, no glutamine (Invitrogen, 11960)
Pen/Strep/Glutamine (Invitrogen 10378-016)
Fetal Bovine Serum (Atlanta Biological, S10350)

Transfection Media
DMEM high glucose, no glutamine (Invitrogen, 11960)
Pen/Strep/Glutamine (Invitrogen 10378-016)

Assay Media
DMEM no phenol red (Invitrogen, 21063-029)
Pen/Strep/Glutamine (Invitrogen 10378-016)
NuSerum (BD biosciences, 24883)

0.05% Trypsin-EDTA (Invitrogen, 25300-062)
Aurora 1536-well Mako plate (Aurora/Brooks, 00028778)

Comment:

External ID: EPAC2-agonist-p1

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: 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 activators are ranked higher than compounds that showed apparent inhibition.
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: EPAC2-antagonist-p1

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 ATA and DMSO.

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: EPAC1-antagonist-p1

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: 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: AMA1100

Protocol: Two microliters of his-tagged AMA1 3D7 allele protein (final concentration 25 nM) was dispensed into a 1,536-well assay plate. Small molecules and positive control peptides were pin-transferred (23 nL or 46 nL) via Kalypsys pin-tool equipped with a 1,536-pin array, resulting in final compound and peptide concentration ranges of 91.5 nM - 57.2 muM, and 15.6 nM - 2.00 muM, respectively. Unlabeled RON2 peptide or R1 peptide (VFAEFLPLFSKFGSRMHILK) that specifically binds the 3D7 AMA1 was used as a positive control that inhibited the binding of RON2L to AMA1. After 15 minutes incubation, 1 uL of biotinylated RON2 peptide (final concentration 25 nM) or buffer were dispensed and the assay plate was incubated for an additional 30 minutes at room temperature and protected from light. This was followed by an addition of 1 uL mixture of donor and acceptor beads (10 ug/mL final concentrations for each). The plates were incubated for 30 min at room temperature and read using the EnVision(R) plate reader (PerkinElmer). Maximum inhibition response was normalized to the positive control signal while no inhibition response was normalized to the DMSO treated wells.

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: ABHD4_INH_FP_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of alpha/beta hydrolase domain containing 4 (ABHD4). In this assay, ABHD4 protein is incubated with test compounds and fluorophosphonate-rhodamine (FP-Rh) activity-based probe. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as ABHD4 inhibitors will prevent ABHD4-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a nominal test concentration of 8.92 micromolar.

Protocol Summary:

Prior to the start of the assay, 4 microliters of assay buffer (50 mM HEPES pH 7.0, 150 mM NaCl and 0.01% Pluronic F-127) were dispensed into column 2 only. Next, 4 microliters of assay buffer containing 46.9 ug/mL of ABHD4 mutant protein (S159A) were dispensed into columns 1 and 3 of 1536 microtiter plates and 4 microliters of assay buffer containing 46.9 ug/mL of ABHD4 wild type protein were dispensed into columns 4 thru 48. Then, 45 nL of test compound in DMSO or DMSO alone (0.89% final concentration) were added to the appropriate wells and incubated for 45 minutes at 25 degrees Celsius.

The assay was started by dispensing 1.0 microliter of 188 nM FP-Rh in assay buffer to all wells. Plates were centrifuged and after 60 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525nm, emission = 598nm) for 30 seconds for each polarization plane (parallel and perpendicular).

Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):

FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )

Where:

Raw1 is defined as the S channel.
Raw2 is defined as the P channel.

The percent inhibition for each compound was calculated as follows:

100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

Test_Compound is defined as wells containing ABHD4 wild type protein in the presence of test compound and FP-Rh.
High_Control is defined as wells containing ABHD4 mutant protein, DMSO and FP-Rh.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:


A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-6, for inactive 6-0.

List of Reagents:

mABHD4 wild type protein (supplied by Assay Provider)
mABHD4 (S159A) mutant protein (supplied by Assay Provider).
FP-Rh probe (supplied by Assay Provider)
HEPES (Sigma, part 83264)
NaCl (Sigma, part S6546)
Pluronic F-127 (Invitrogen, part P6866)
1536-well plates (Corning, part 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: COUPTFII_INH_LUMI_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this cell-based assay is to identify compounds that can inhibit COUP-TFII transcriptional activity. The expression vectors for COUP-TFII (pcDNA6.2-COUP-TFII) and the luciferase reporter NGFIA-Luc (pXP2-168) are transiently cotransfected into HEK-293T cells.

COUP-TFII has been shown to efficiently activate NGFI-A-Luc expression (17) and the readout can be measured by a luminometer. Small molecules that inhibit COUP-TFII transcriptional activity will decrease the promoter activity that can be detected by luciferase assay. As designed, compounds that inhibit COUP-TFII activity will decrease luciferase activity, resulting in decreased well luminescence.

Protocol Summary:

HEK293 cells were routinely cultured in T-175 flasks containing 25 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v antibiotic-antimycotic mix at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, the HEK293 cells were harvested using 5 mL of TrypLE reagents and seeded in fresh media at a density of 10 million cells per T175 flask. The following day, cells were transfected with 1 mL of serum-free OptiMEM containing 12 ug of the plasmid encoding for COUP-TF2, 8 ug of the pX2-168 luciferase reporter plasmid, and 60 uL of X-tremeGene 9 transfection reagent. Twenty four hours post transfection, cells were harvested using 5 mL of preheated TrypLE and resuspended at a concentration of 800,000 cells per mL in phenol-red free DMEM supplemented as above. In the absence of a pharmacological positive control, COUP-TF2 inhibition was mimicked by transfecting cells in absence of the COUP-TF2 expressing vector (i.e. reporter plasmid only).

The assay was started by dispensing 5 uL of cell suspension into each well of white, sterile, solid-bottom 1536-well plates. The first three columns received control cells (no COUP-TF2 expressed) whereas the rest of the plate was dispensed with COUP-TF2-transfected cells. After addition of cells the plates were spun down. The plates were incubated for 4 hours and then treated with 43 nL/well of test compounds or DMSO (final concentration 0.65%) on COUP-TF2 cells and Control cells. Plates were incubated for eighteen hours at 37 C, 5% CO2 and 95% RH. Plates were then removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by addition of 5 uL of One-Glo reagent to each well. After a 10 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer).

The percent inhibition for each compound was calculated as follows:

%_Inhibition = ( ( Test_Compound - Median_Sample_Field ) / ( Median_High_Control - Median_Sample_Field ) ) * 100

Where:

High_Control is defined as wells containing cells transfected with reporter plamid only (pX2-168)
Test_Compound is defined as well containing cells cotransfected with pcDNA6.2-COUP-TFII and pX2-168 in the presence of test compounds
Sample_Field is defined as wells containing cells cotransfected with pcDNA6.2-COUP-TFII and pX2-168 in the presence of test compounds

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells that are within the High and Low Controls (i.e. higher than the average plues three standard deveiations of the Low Control wells and lower than the average minus three stardard deviation of the High Control wells) and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-34, and for inactive compounds 34-0.

List of Reagents:

pX2-168 luciferase reporter plasmid (Assay Provider)
pcDNA6.2-COUP-TFII plasmid (Assay Provider)
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 21063)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
X-tremeGENE 9 DNA Transfection Reagent (Roche, part 06365809001)
OptiMEM (Invitrogen, part 31985)
TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)
OneGlo (Promega, part E6130)
1536-well plates (Corning, part 7298)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: UNMCMD_Botulinum_LightChainF_Protease_HTS_MLPCN

Protocol: This assay will be used to identify small molecule inhibitors of Botulinum toxin type A and F light chain proteases (BoNTALC and BoNTFLC) and inhibitors of Bacillus anthracis Lethal Factor protease (LF) in a multiplex format. Proteases that are not inhibited will cleave their substrates and produce a loss of signal effect. If the proteases are inhibited by a small molecule cleavage will not occur, thus the signal should stay the same. Full length protease substrates are used in order to detect inhibitors that act at either the protease catalytic site or at sites of protease interaction with substrate distal to the cleavage site.

Protease inhibition assays were performed as previously described (5), but with modifications. Biotinylated GFP protease substrates for LF, BoNTALC, BoNTFLC, and a protease-resistant substrate (pinpointGFP) were prepared and loaded on color-coded streptavidin microspheres (Spherotech Blue Array Particle kit, 5.1 microm diameter) as previously described (5-7). Additions to wells were in sequence as follows: 1st, 4 microL protease buffer (50 mM HEPES, 100 mM NaCl, 1 mg/ml bovine serum albumin, 0.025% Tween-20, pH 7.4); 2nd, 2 microL of a mixture of 1.5 microM LF, 5 nM BoNTALC and 375 nM BoNTFLC in protease buffer; 3rd, 100 nL of test compounds (1 mM in DMSO); and 4th, 4 microL containing 2x10;5/ml of each set of microspheres. Plates were sealed and incubated at 24 degreesC overnight (16-18 h), rotating continuously from inverted to upright position until analyzed in 1536 well plate format with the HyperCyt Cluster Cytometer platform the following day.

The assay response range was defined by replicate control wells containing protease buffer alone (PCntrl, positive control) or the protease mixture alone (NCntrl, negative control). Additional positive controls included Ebselen, VAMP peptide and IN-2-LF (selective inhibitors of BoNTALC, BoNTFLC and LF, respectively), which were added separately and together as a mixture to validate protease inhibition. In each well the median fluorescence intensity (MFI) was determined for each set of substrate-bearing microspheres. A fifth streptavidin microsphere set included in each well had no GFP moiety attached and was used to quantify the contribution of innate test compound fluorescence to the assay readout. All MFI values for substrate-bearing microspheres were corrected by subtraction of the MFI value for the substrate-free microspheres in the same well.

Test compound inhibition of substrate cleavage by protease was then calculated as 100 x (MFI:Test - Mean MFI:NCntrl)/(Mean MFI:PCntrl - Mean MFI:NCntrl) in which MFI:Test represents corrected MFI in the
presence of test compound, and Mean MFI:PCntrl and MFI:NCntrl represent means for replicate MFI determinations in control wells.

PubChem Score equals percent inhibition.

Comment: Development of the HyperCyt Cluster Cytometer Platform for processing of 1536 well plates by high throughput flow cytometry was supported by NIH/NIMH Grant 1R01HG005066 to Bruce Edwards.

External ID: 2152-01_Activator_SinglePoint_HTS_Activity

Protocol: . Coat plates with 0.1% Gelatin in PBS (20ul per well) for 20 minutes at room temp.
Day 0
. Plate 3,000 HUVEC cells/well in 384 well plates, 50l per well, in EGM-2 medium, incubate at 37 degrees Celsius
Day 2
. Pin 100nl of compound, DMSO
. Add 10 ul Positive Control (TNFalpha at 0.3ng/ml)
. Incubate 16h
Day 3
. Aspirate media using plate washer
. Add 70 ul wash buffer using Thermo Multidrop Combi or plate washer (assay provider uses combi)
. Aspirate wash buffer with plate washer
. Add 20 ul blocking antibody (IgG) with combi
. Incubate at room temp for 15 minutes
. Aspirate
. Add 20 ul Biotin conjugated anti-e-selectin antibody with combi
. Incubate 30 minutes at room temp (or 15 minutes at 37 degrees Celsius)
. Aspirate
. Add 70 ul wash buffer using combi or plate washer (assay provider uses combi)
. Aspirate
. Add 20 ul streptavidin-PE using combi or plate washer (assay provider uses combi)
. Incubate 30 minutes at room temp (or 15 minutes at 37 degrees Celsius)
. Aspirate
. Add 70 ul of PBS (not wash buffer)
. Aspirate
. Add 20ul Hoechst/4% PF/PBS mix (fix cells and stain nuclei)
. Incubate 15 minutes at room temp
. Aspirate (PF is a hazardous waste and is collected in a seperate hazardous waste container)
. Add 70ul PBS
. Aspirate
. Add 70 ul PBS
Plates are sealed with foil seal and stored overnight at 4 degrees Celsius prior to imaging

Plates are imaged on an automated microscope (ImageXpress Micro, Molecular Devices Inc.) at 2x magnification using appropriate filter sets for Hoechst and PE fluorescence.

Cell counts and % of cells positive for PE fluorescence are quantified by using a modified Cell Scoring application module in the image analysis software (MetaXpress, Molecular Devices Inc.)

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
Compounds decreasing cell viability by more than 20 per cent were eliminated. The minimum PUBCHEM_ACTIVITY_SCORE of E-selectin positive cells required for a compound to be called a hit (the activity threshold, or AT) was set at 10.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 60.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: UNMCMD_Botulinum_LightChainA_Protease_HTS_MLPCN

Protocol:
This assay will be used to identify small molecule inhibitors of Botulinum toxin type A and F light chain proteases (BoNTALC and BoNTFLC) and inhibitors of Bacillus anthracis Lethal Factor protease (LF) in a multiplex format. Proteases that are not inhibited will cleave their substrates and produce a loss of signal effect. If the proteases are inhibited by a small molecule cleavage will not occur, thus the signal should stay the same. Full length protease substrates are used in order to detect inhibitors that act at either the protease catalytic site or at sites of protease interaction with substrate distal to the cleavage site.

Protease inhibition assays were performed as previously described (5), but with modifications. Biotinylated GFP protease substrates for LF, BoNTALC, BoNTFLC, and a protease-resistant substrate (pinpointGFP) were prepared and loaded on color-coded streptavidin microspheres (Spherotech Blue Array Particle kit, 5.1 microm diameter) as previously described (5-7). Additions to wells were in sequence as follows: 1st, 4 microL protease buffer (50 mM HEPES, 100 mM NaCl, 1 mg/ml bovine serum albumin, 0.025% Tween-20, pH 7.4); 2nd, 2 microL of a mixture of 1.5 microM LF, 5 nM BoNTALC and 375 nM BoNTFLC in protease buffer; 3rd, 100 nanoL of test compounds (1 mM in DMSO); and 4th, 4 microL containing 2x10;5/ml of each set of microspheres. Plates were sealed and incubated at 24 degreesC overnight (16-18 h), rotating continuously from inverted to upright position until analyzed in 1536 well plate format with the HyperCyt Cluster Cytometer platform the following day.

The assay response range was defined by replicate control wells containing protease buffer alone (PCntrl, positive control) or the protease mixture alone (NCntrl, negative control). Additional positive controls included Ebselen, VAMP peptide and IN-2-LF (selective inhibitors of BoNTALC, BoNTFLC and LF, respectively), which were added separately and together as a mixture to validate protease inhibition. In each well the median fluorescence intensity (MFI) was determined for each set of substrate-bearing microspheres. A fifth streptavidin microsphere set included in each well had no GFP moiety attached and was used to quantify the contribution of innate test compound fluorescence to the assay readout. All MFI values for substrate-bearing microspheres were corrected by subtraction of the MFI value for the substrate-free microspheres in the same well.

Test compound inhibition of substrate cleavage by protease was then calculated as 100 x (MFI:Test - Mean MFI:NCntrl)/(Mean MFI:PCntrl - Mean MFI:NCntrl) in which MFI:Test represents corrected MFI in the
presence of test compound, and Mean MFI:PCntrl and MFI:NCntrl represent means for replicate MFI determinations in control wells.

PubChem Score equals the percent inhibition.

Comment: Development of the HyperCyt Cluster Cytometer Platform for processing of 1536 well plates by high throughput flow cytometry was supported by NIH/NIMH Grant 1R01HG005066 to Bruce Edwards.

External ID: SIAE_INH_FP_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of sialic acid acetylesterase (SIAE). In this assay, SIAE protein is incubated with test compounds and fluorophosphonate-rhodamine (FP-Rh) activity-based probe. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as SIAE inhibitors will prevent SIAE-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a nominal test concentration of 9.66 micromolar.

Protocol Summary:

Prior to the start of the assay, 3 microliters of assay buffer (1X DPBS and 0.01% Pluronic F-127) were dispensed into column 1 thru column 3 of 1536 microtiter plates. Next, 3 microliters of assay buffer containing 0.73uM of SIAE protein were dispensed into columns 4 thru 48. Then, 39 nL of test compound in DMSO or DMSO alone (0.97% final concentration) were added to the appropriate wells and incubated for 45 minutes at 25 degrees Celsius.

The assay was started by dispensing 1.0 microliter of 300 nM FP-Rh in assay buffer to all wells. Plates were centrifuged and after 120 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525nm, emission = 598nm) for 25 seconds for each polarization plane (parallel and perpendicular).

Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):

FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )

Where:

Raw1 is defined as the S channel.
Raw2 is defined as the P channel.

The percent inhibition for each compound was calculated as follows:

100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

Test_Compound is defined as wells containing SIAE protein in the presence of test compound and FP-Rh.
High_Control is defined as wells containing DMSO, FP-Rh but, no SIAE protein.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-2, for inactive 2-0.

List of Reagents:

SIAE protein (supplied by Assay Provider)
FP-Rh probe (supplied by Assay Provider)
DPBS (Mediatech, part 20-031-CV)
Pluronic F-127 (Invitrogen, part P6866)
1536-well plates (Greiner, part 789176)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: UNMCMD_Anthrax_LethalFactor_Protease_HTS_MLPCN

Protocol: This assay will be used to identify small molecule inhibitors of Botulinum toxin type A and F light chain proteases (BoNTALC and BoNTFLC) and inhibitors of Bacillus anthracis Lethal Factor protease (LF) in a multiplex format. Proteases that are not inhibited will cleave their substrates and produce a loss of signal effect. If the proteases are inhibited by a small molecule cleavage will not occur, thus the signal should stay the same. Full length protease substrates are used in order to detect inhibitors that act at either the protease catalytic site or at sites of protease interaction with substrate distal to the cleavage site.

Protease inhibition assays were performed as previously described (5), but with modifications. Biotinylated GFP protease substrates for LF, BoNTALC, BoNTFLC, and a protease-resistant substrate (pinpointGFP) were prepared and loaded on color-coded streptavidin microspheres (Spherotech Blue Array Particle kit, 5.1 microm diameter) as previously described (5-7). Additions to wells were in sequence as follows: 1st, 4 microL protease buffer (50 mM HEPES, 100 mM NaCl, 1 mg/ml bovine serum albumin, 0.025% Tween-20, pH 7.4); 2nd, 2 microL of a mixture of 1.5 microM LF, 5 nM BoNTALC and 375 nM BoNTFLC in protease buffer; 3rd, 100 nL of test compounds (1 mM in DMSO); and 4th, 4 microL containing 2x10;5/ml of each set of microspheres. Plates were sealed and incubated at 24 degreesC overnight (16-18 h), rotating continuously from inverted to upright position until analyzed in 1536 well plate format with the HyperCyt Cluster Cytometer platform the following day.

The assay response range was defined by replicate control wells containing protease buffer alone (PCntrl, positive control) or the protease mixture alone (NCntrl, negative control). Additional positive controls included Ebselen, VAMP peptide and IN-2-LF (selective inhibitors of BoNTALC, BoNTFLC and LF, respectively), which were added separately and together as a mixture to validate protease inhibition. In each well the median fluorescence intensity (MFI) was determined for each set of substrate-bearing microspheres. A fifth streptavidin microsphere set included in each well had no GFP moiety attached and was used to quantify the contribution of innate test compound fluorescence to the assay readout. All MFI values for substrate-bearing microspheres were corrected by subtraction of the MFI value for the substrate-free microspheres in the same well.

Test compound inhibition of substrate cleavage by protease was then calculated as 100 x (MFI:Test - Mean MFI:NCntrl)/(Mean MFI:PCntrl - Mean MFI:NCntrl) in which MFI:Test represents corrected MFI in the
presence of test compound, and Mean MFI:PCntrl and MFI:NCntrl represent means for replicate MFI determinations in control wells.

Pubchem Activity Score equals the percent compound inhibition.

Comment: Development of the HyperCyt Cluster Cytometer Platform for processing of 1536 well plates by high throughput flow cytometry was supported by NIH/NIMH Grant 1R01HG005066 to Bruce Edwards.

External ID: PRPC_INH_TRFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as inhibitors of PRPc cell surface expression. This assay detects PrPc on the surface of living LD9 fibroblast cells using two PRPC-specific antibodies which carry donor and acceptor fluorophores, respectively.

In this assay, the antibodies are directed against distinct, non-overlapping PrPc epitopes. Antibody occupancy of both epitopes of a PrPc molecule results in FRET signal emission. Free antibody or single antibody occupancy is not detected. The assay employs long half-life lanthanide donors (Europium or Terbium cryptate) and acceptor fluorophores XL665 or d2: SAF32 (aa53-93) and D18 (aa133-157) labeled with the donor and acceptor fluorophores, respectively. Ratios of 665 nm to 620 nm measurements are calculated, to take into account non-specific absorption of 620 nm light by the assay matrix. Compounds are tested in singlicate at a final nominal concentration of 13.8 microM.

Protocol Summary:

The LD9 cell line (subclone of L929, which is a mouse fibroblast cell line) was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Opti-MEM I Reduced Serum Medium (no phenol red) supplemented with 5% v/v Bovine Growth Serum Supplemented Calf and 1X Penicillin-Streptomycin.

The day before the assay, 3 uL of cell growth media was dispensed into the first two columns of 1536 well microtiter plates and 1500 cells in 3 uL of cell growth media were seeded into the remaining wells. Next, 42 nL of test compound in DMSO, Brefeldin A (58 uM final concentration) in DMSO, or DMSO alone (1.38% final concentration) were dispensed to the appropriate wells. The plates were then incubated for 21 hours at 37 C, 5% CO2, and 95 % RH.

The assay was started by adding 1 uL of SAF32-Tb at a final concentration of 0.072ug/mL and 1ul of D18-d2 at a final concentration of 0.66ug/mL to all wells, respectively. Labeled Antibodies were prepared in 1X PBS. Plates were centrifuged and after 3 hours of incubation at room temperature, well TRFRET was read on a ViewLux microplate reader (Perkin Elmer, Turku, Finland). Measurements were performed by exciting the plates at 340 nM, and monitoring well fluorescence at 618 nm (Tb) and 671 nm (d2) with the ViewLux microplate reader.

To normalize data, values measured from both fluorescence emission wavelengths were used to calculate a ratio for each well, according to the following mathematical expression:

Ratio = I671nm / I618nm

Where:

I represents the measured fluorescence emission intensity at the enumerated wavelength in nm.

The percent inhibition was calculated from the median ratio as follows:

%_Inhibition = 100 * ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) )

Where:

Test_Compound is defined as wells containing cells, test compounds and DMSO.
High_Control is defined as wells containing cells, Brefeldin A and DMSO.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-18, and for inactive compounds 18-0.

List of Reagents:

LD9 cells (supplied by Assay Provider)
Opti-MEM Reduced Serum Medium (Life Technologies, part 11058-021)
Bovine Growth Serum Supplemented Calf (Hyclone, part SH30541.03)
Penicillin-Streptomycin (100X) (Life Technologies, part 15140-122)
Brefeldin A (Control, Sigma, part B7651)
SAF32-Tb (Cisbio, custom labeled antibody)
D18-d2 (Cisbio, custom labeled antibody)
PBS (Life Technologies, part 10010-031)
Detachin Cell Detachment Reagent (Genlantis, part T100100)
T-175 Flasks (Nunc, part 159910)
1536-well plates (Corning, part 7298)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: 7052-01_Inhibitor_SinglePoint_HTS_Activity_Set2

Protocol: Caspase 6 enzyme was received from collaborator and stored at -80 C. Stock concentration was at 1.2 mg/ml (43.1 uM).

Assay Buffer:
100 mM Hepes pH 7.5, 10% sucrose, 0.1 % CHAPS, 5 mM DTT (final concentration) was added fresh just prior to assay

Reagents:
VEID-R110-VEID: Caspase 6 substrate, was reconstituted to 10 mM from dry powder with DMSO and stored at -20 C.
VEID-CHO: Caspase inhibitor (positive control) was reconstituted to 10 mM from dry powder with DMSO and stored at -20 C.

Assay Ready Plates:
1536 black, solid bottom plates containing 7.5 ul of compound

Protocol:
Add 4 ul 2.2 nM Caspase-6 to 1536 ARPS using BioRaptR
Add 100 nL VEID-CHO to positive control wells using Combi nL
Incubate Room temp 60 min
Add 2 ul of 24 uM VEID-R110-VEID (Caspase 6 substrate) to all wells
Incubate at room temp for 40 min
Read plate on Envision (FITC settings and filters)

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 35.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: UHRF1-CPGDNA_INH_TRFRET_1536_1X%INH CSRUN for MBD2-CPGDNA INH

Protocol: Assay Overview:

The purpose of this assay is to determine whether compounds identified as inhibitors of MBD2 are nonselective, as determined by inhibition of methylated DNA-binding activity of the SRA domain polypeptide of ubiquitin-like with PHD and ring finger domains 1 (UHRF1). The UHRF1 gene encodes a member of a subfamily of RING-finger type E3 ubiquitin ligases. It contains a Set-and RING Associated (SRA) domain that binds to partially methylated DNA sequences during DNA synthesis. UHRF1 exhibits a different mode of DNA binding than MBD2, binding so-called hemi-methylated cytosines in order to promote their fully methylation. This assay is performed in a similar manner as the MBD2 screen, except that it employs recombinant UHRF1 SRA domain protein (UHRF1_SRA) and hemi-methylated DNA as its substrate. This assay monitors the ability of compounds to inhibit binding of cloned recombinant UHRF1_SRA to a FAM-labeled 14 bp hairpin oligonucleotide containing a hemimethylated CpG. In this biochemical assay, test compounds are incubated with UHRF1_SRA and a 5-carboxyfluorescene (FAM)-labeled 14bp hairpin DNA oligonucleotide containing one hemimethylated CpG dinucleotide in the presence of Tb-labeled anti-His antibody. An inhibitor of the UHRF1_SRA:FAM hemimethylated CpG interaction will inhibit binding between UHRF1 and FAM hemimethylated oligo, leading to reduced energy transfer and reduced well FRET (fluorescein emission / Tb emission). Compounds are tested in singlicate at a final nominal concentration of 4.4 micromolar.

Protocol Summary:

The assay was started by dispensing 5 microliters of Control Mix in assay buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 125 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.2% Tween-20) containing 125 nanomolar of UHRF1 protein, 5 micromolar of FAM labeled methylated CpG DNA oligo and 5 nanomolar of Terbium labeled anti-his antibody into columns 1 thru columns 2 of 1536 microtiter plates. Next, 5 microliters of Experimental Mix containing 125 nanomolar of UHRF1 protein, 100 nanomolar FAM labeled Hemimethylated CpG DNA oligo and 5 nanomolar Terbium labeled anti-his antibody in assay buffer were dispensed into columns 3 thru 48. Then, the plates were centrifuged and pinned with 22 nL of test compound in DMSO, Mitoxantrone (1.3 micromolar final concentration) in DMSO or DMSO alone (0.44% final concentration). The plates were incubated for 60 minutes at 25 degrees Celsius and TR-FRET was measured on a Viewlux microplate reader (Perkin Elmer, Turku, Finland). After excitation at 340 nm, well fluorescence was monitored at 495 nm (Tb) and 525 nm (FAM). For each well, a fluorescence ratio was calculated according to the following mathematical expression:
Ratio = I525nm / I495nm

Where:

I525nm represents the measured fluorescence emission at 525 nm.
I495nm represents the measured fluorescence emission at 495 nm.

The percent inhibition for each compound was calculated using as follows:

%_Inhibition = 100 * ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) )

Where:

Test_Compound is defined as wells containing the Experimental Mix in the presence of test compound.
High_Control is defined as wells containing the Experimental Mix, Mitoxantrone and DMSO.
Low_Control is defined as the median of the wells containing test compounds.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported Pubchem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-19, and for inactive compounds 19-0.

List of Reagents:

UHRF1_SRA protein (supplied by Assay Provider)
FAM labeled Hemimethylated CpG DNA Oligo (IDT-custom order)
FAM labeled Methylated CpG DNA Oligo (IDT-custom order)
Lanthascreen Tb Anti-His Antibody (Invitrogen, part PV5895)
MgCl2 (Fisher, part BP214)
NaCl (Sigma, part S6546)
DTT (Fisher, part BP172)
EDTA (Sigma, part E7889)
Tris (Amresco, part 0497)
Tween 20 (Sigma, part P9416)
Glycerol (Sigma, part G7893)
1536-well plates (Corning, part 7261)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: DAX1-FULL_INH_LUMI_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that can reduce DAX-1 activity. DAX-1 has been shown to inhibit SF-1-mediated transactivation (5).

SF-1 (steroidogenic factor 1; NR5A1) has been shown to function as a transcription factor for a variety of different steroidogenic enzyme genes in the adrenal gland and gonads. Specifically, SF-1 has been shown to induce differentiation of mesenchymal stem cells into steroidogenic cells with concomitant strong induction of StAR expression.

This assay employs HEK293T cells co-transfected with plasmids expressing SF-1 and a luciferase reporter under control of the StAR promoter together with an expression plasmids for DAX-1, followed by treatment with test compounds. Under normal conditions, DAX-1 represses the SF-1-induced expression of the luciferase reporter. As designed, a compound that inhibits DAX-1 activity will reduce DAX1-mediated repression of SF-1, thereby allowing SF-1 to increase StAR promoter activity, resulting in increased well luminescence. Compounds will be tested in singlicate at a nominal concentration of 6.8 uM.

Protocol Summary:

HEK293 cells were routinely cultured in T-175 flasks containing 25 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v antibiotic-antimycotic mix at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, the HEK293 cells were harvested using 5 mL of TrypLE reagents and seeded in fresh media at a density of 10 million cells per T175 flask. The following day, cells were transfected with 5 mL of serum-free OptiMEM containing 28 ug of the StAR promoter luciferase reporter plasmid, 14 ug of the SF-1 expressing palsmid, 21 ug of the DAX-1 expressing plasmid and 100 uL of transfection reagent. Four hours post transfection, cells were harvested using 5 mL of preheated TrypLE and resuspended at a concentration of 800,000 cells per mL in phenol-red free DMEM supplemented as above. In the absence of a pharmacological positive control, DAX-1 inhibition was mimicked by transfecting cells with an empty vector in place of the DAX-1 expressing vector.

The assay was started by dispensing 5 uL of cell suspension into each well of white, solid-bottom 1536-well plates using a flying reagent dispenser (Aurora). The first three columns received control cells (no DAX-1 expressed) whereas the rest of the plate was dispensed with DAX1-transfected cells. The plates were then treated with 34 nL/well of test compounds or DMSO (final concentration 0.68%) on DAX-1 cells and Control cells using a PinTool transfer unit (GNF). Plates were incubated for eighteen hours at 37 C, 5% CO2 and 95%RH. Plates were then removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by addition of 5 uL of SteadyLite reagent to each well. After a 15 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer).

The percent inhibition of each test compound was calculated as follows:

%_Inhibition = ( 1 - ( median_positive_control - test_compound ) / (median_positive_control - median_negative_control ) * 100

Where:

Positive control is defined as wells containing control cells (no DAX-1 expressed) treated with DMSO.
Negative control is defined as wells containing DAX-1 cells treated with DMSO.
Test compound is defined as wells containing DAX-1 cells containing test compound.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally active compounds. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-4, and for inactive compounds 4-0.

List of Reagents:

pGL2_1.3 kb_StAR luciferase reporter plasmid (Assay Provider)
pSG.SF-1 plasmid (Assay Provider)
pSV.DAX-1 plasmid (Assay Provider)
pSG5 empty vector (Assay Provider)
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 11965)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
TransIT 293 (Mirus Corporation, part MIR-2700)
OptiMEM (Invitrogen, part 31985)
TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)
SteadyLite Reagent (PerkinElmer, part 6016989)
1536-well plates (Greiner part 789173)

Comment: Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

External ID: SBCCG-A1021-TROY-Primary-Assay

Protocol: Q293 NFkB-Luc LMP1-TROYcd cell-based assay in 1536
Materials:
1.#Q293 LMP1 TROYcd NF-kappaB Luc Cell line
2.#DMEM (Invitrogen Catalog # 11965-118)
3.#FBS (Invitrogen Catalog # 16000-044)
4.#0.05% Trypsin (Invitrogen Catalog # 25300-054)
5.#Puromycin
6.#Opti-MEM (Invitrogen Catalog # 11058)
7.#Flat Bottom White Solid Bottom Tissue Culture Treated 384-Well Plate (Corning 3750)
8.#PBS (Invitrogen Catalog # 10010-049)
9.#Bright-Glo Reagent Kit (Promega Catalog # 2610)

Tissue culture:
1.#Growth Media: DMEM/10%FBS/1X L-Glu/1X PenStrep/ 3 ug/ml Puromycin
2.#Seeding Media: Opti-MEM/1X L-Glu/1X PenStrep ( make fresh media right before seeding)
3.#1536 plate: Corning 3727 (White, solid, TC treated)

HTS Procedure:
a.#Harvest and seed cells: 1000 c/well, 4 ul/well (or 250,000 c/ml)
b.#Add 4 ul/well of sedding media to cols 1-4
(Note: I saw cross talk in col 4)
c.#Add 4 ul/well of cells to cols 5-48
d.#After seeding, spin down the plates for 1 min, 1000 rpm
e.#Incubate the plates in the incubator (37C/5%CO2) for 24 hours ( +/- 2 hours)
f.# Add DMSO to cols 1-8, and compounds to cols 9-48 using ECHO
g.#Incubate the plates in the incubator (37C/5%CO2) for 4 hours
h.#Take the plates out to equilibrate the plates at RT for 10 min
(Note: based on my observation, equilibrate the plates at RT helps to avoid the edge effect)
i.#Add 4 ul/well of Bright-Glo
j.#Incubate the plates at RT for 5 minutes
k.#Read the plates on ViewLux , using protocol: WO7_LUMI_corning1536_02262013

Comment: The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.

In our data, the "Value at 10 uM" column refers to the raw value as measured by the plate reader. The "%Activity_Corrected at 10 uM" column refers to the same raw value that has been normalized and corrected using the Genedata software. Active compounds are those that satisfy:

"%Activity_Corrected at 10 uM" >= 50%

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.

Activity Scoring

Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:

1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:

a. If outcome of the primary screen is inactive, then the assigned score is 0

b. If outcome of the primary screen is inconclusive, then the assigned score is 10

c. If outcome of the primary screen is active, then the assigned score is 20

Scoring for Single concentration confirmation screening is not applicable to this assay.

d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21

e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25

f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30

This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay

External ID: SBCCG-A1016-RevErbaLBD-Primary-Assay

Protocol: This assay is to identify modulators of Rev-erb alpha protein binding to DNA

A. Materials:
REV-alpha_beta purified protein = Rastinejad lab
FITC-DNA = Rastinejad lab
Tris = Biorad (cat #161-0719)
DTT = Akron Biotechnology (cat #AK2948-0005)
5M NaCl = Sigma-Aldrich (cat #56546-1L)
Glycerol 99.5% = Acros (cat #327255000)
Tween 20 = Sigma-Aldrich (cat #P1379)
Corning high base black plates = Corning (cat #3724)
Molecular grade water = Cellgro (cat #46-000-CM)

B. Plate Map:
Negative (low) control in columns 1 and 2 is 6nM DNA and 35nM Protein, DMSO
Positive (high) control in columns 3 and 4, Protein at 750nM and 6nM DNA, DMSO
Test compound in columns 5 - 48, Protein at 35nM + 6nM DNA + test compound

C. Procedures:
Step#Description
1#Prepare 2X Rev-erb alpha protein stock and 2X DNA stock
2#Using LabCyte Echo, transfer xnL from a 2 mM Echo qualified plate containing test compounds into assay plate Col. 5 - 48. Add same volume of DMSO in col 1-4.
3#Spin plates at 1000 rpm for 1 minute in centrifuge.
4#Using the bioraptr, add 3 uL/well of (35nM protein control) to columns 1 and 2 and test compound wells.
5#Using the bioraptr, add 3 uL/well of Mix 2 (750nM protein) to col. 3-4 for the positive control
6#Using the bioraptr, add 3uL/well of Mix 3 (DNA) to col. 1-48.
7#Spin plates at 1000 rpm for 1 minute in centrifuge.
8#Incubate plates in the dark at room temperature for 90 minutes.
9#Set up Perkin Elmer EnVision as described in section Instrument setting.
10#Read plates on EnVision using FP Dual enhanced mirror, FP 480 excitation filter, FP-P-pol 535 and FP-S-pol 535 emissin filters

Comment: Actives were selected based on, % response = 45% or greater