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: 7056-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: Bac rRNA Automation Protocol
Day 1:
1. Grow each E. coli strain (one wild type (GFP) and six humanized mutants (RFP) including the control G2058) individually. Pick each strain into a separate 50 mL conical tube with 10 mL of LB medium supplemented with 100 ug/mL amplicillin and 50 ug/mL spectinomycin.
2. Incubate strains overnight at 37 degrees with shaking at 190 RPM.
Day 2:
1. In the morning, check OD600 of the cultures.
2. Dilute the cultures to OD 600 = 0.05 in LB medium supplanted with 100 ug/mL ampicillin.
3. Return strains to 37 degrees with shaking at 190 RPM for about 3. 5 hours. Grow strains to reach OD 600 about 0.5 (exponential growth phase).
4. Mix strains (final OD600= 0.02) in the proportion 50% wild-type, 8.33% each of the six mutants in LB medium supplemented with 100 ug/mL ampicillin and 1 mM IPTG (which is used to induce expression of fluorescent proteins).
5. Dispense 30 uL/well of bacteria mixture to 384 well Assay Ready Plates (ARPs) containing positive controls (Erythromycin 15 mg/mL, 50 nL in column 1 and Choramphenicol 15 mg/mL, 50 nL in column 23).
6. Place breathable film on plate using Plate Loc.
7. Incubate plates in Steristore at 37 degrees, 0% CO2 for 24 hours, 95% humidity.
Day 3:
1. Remove assay plate from Steristore.
2. Remove breathable film with X-Peel.
3. Read GFP signal using EnVision plate reader (mirror: FITC #403, Ex: FITC 485 #102, Em: FITC 535 #206).
4. Read RFP signal using EnVision plate reader (mirror: BODIPY TMR #405, Ex: photometric 550 #312, Em: Cy3 595 #229).
5. Read OD600 signal using EnVision plate reader (Ex: Photometric 600 #319).
6. Store plate in Steristore incubator.
Equipment Required
Combi
Plate Loc
Steristore (37 C, 95% humiodity, 0% CO2,)
X-Peel
2 EnVisions

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control
wells (PC) were included on every plate. One positive control,
Chloramphenicol, was used to determine the baseline fluorescence and OD
as it was expected to non-selectively kill all 6 strains, GFP wild-type
and RFP mutant. The other positive control, Erythromycin, was expected
to selectively spare one RFP control strain (G2058) and was therefore
used as a guideline for calculating the performance of a selective GFP
inhibitor.

NORMALIZATION:
Each of three layers was independently normalized using the 'Neutral
Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to
a normalized activity value of 0.
The a raw signal of 0 was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: The plate pattern correction algorithm 'Assay
Pattern (multiplicative)' in Genedata (v7.0.3) was applied to the
normalized plate data for each layer.

AGGREGATE COMPOUND LAYER SCORING: In addition to the reported normalized
percent activities above, each replicate compound measurement was
converted to a z-score relative to the corresponding DMSO-control
distribution, as described for ChemBank (Seiler et al., 2008). ChemBank
composite Z scores were computed to combine replicates for each channel
(RFP, GFP, and OD). We sought compounds that affected the GFP-expressing
wild-type strain while not affecting at least one of the mutant RFP
strains and not affecting overall viability as measured by OD; i.e.,
they score with negative composite Z in the GFP channel with little or
no effect in the RFP or OD channels. To combine the results of the three
channels quantitatively, we defined a trigonometric scaling function to
express our preferences that (1) GFP composite Z scores be negative, and
(2) RFP and OD composite Z score amplitudes exceed GFP composite Z score
amplitudes; such compounds should fall left of the DMSO-control
distribution, but above the main diagonal in a plot of GFP composite Z
versus either OD or RFP composite Z. Using the two ratios of ChemBank
composite Z scores (GFP/RFP and GFP/OD), we defined q1 = arctan(GFP/RFP)
and q2 = arctan(GFP/OD) and expressed our scale factor as F =
cos(-q1)cos(2q1)cos(-q2)cos(2q2), with the four factors respectively
expressing our preferences. As a three-assay composite score, we take
-(ZGFP) x F, which clearly discriminates the compounds of interest when
plotted against RFP or OD.

PUBCHEM_ACTIVITY_SCORE: The above three-assay composite score was
normalized on a scale of 1-100, with 1 being those with the least
desirable observed score representing compounds increasing in GFP signal
and decreasing in RFP or OD, and 100 being the most ideal observed score
representing a loss in GFP with no change in RFP or OD.

PUBCHEM_ACTIVITY_OUTCOME: The above three-assay composite scores were
fit to a standard distribution and a corresponding Holm-Bonferroni
corrected p value significance was calculated. Compounds with a FDR
corrected p value less than 0.05, normalized OD percent score > -30,
negative GFP composite Z score, and the expression Abs(Cos(-q)Cos(2q)) >
(sqrt2)/2 for both q1 and q2 were assigned a score of 2 (active).
Compounds failing any of these criteria were assigned a score of 1
(inactive.)

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: HIV Cellular Data

Protocol: N/A

Comment: N/A

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: VP16_INH_LUMI_1536_3X%INH CSRUN for COUP-TFII (NR2F2)

Protocol: Assay Overview:

The purpose of this assay is to determine whether the hits identified as active in a set of experiments entitled, Luminescence-based cell-based primary high throughput screening assay to identify antagonists of COUP-TFII (NR2F2) (AID 686940), are assay artifacts affecting luciferase activity, cell viability or the transcription/translation machinery in a non-selective manner.


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 9.9 ug of the plasmid encoding VP16 (pAB-Gal4-VP16), 9.9 ug of the p17 mer x4-TK-Luc luciferase reporter plasmid, and 65 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, VP16 inhibition was mimicked by transfecting cells in absence of the VP16 expressing vector (i.e. reporter plasmid only). Compounds are tested in triplicate at a final nominal concentration of 8.5 uM.

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 VP16 expressed) whereas the rest of the plate was dispensed with VP16-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_Low_Control ) / ( Median_High_Control - Median_Control_Field ) ) * 100

Where:
Low_Control is defined as wells
High_Control is defined as wells containing cells transfected with reporter plamid only (p17 mer x4-TK-Luc)
Test_Compound is defined as well containing cells cotransfected with pAB-Gal4-VP16 and p17 mer x4-TK-Luc in the presence of test compounds

PubChem Activity Outcome and Score:

The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the primary screen (AID 686940) 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 100-25, and for inactive compounds 24-0.

List of Reagents:

p17 mer x4-TK-Luc luciferase reporter plasmid (Assay Provider)
pAB-Gal4-VP16 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. The MLSMR was not able to provide all compounds selected for testing in this assay.

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: 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: 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-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

External ID: VP16_INH_LUMI_1536_3XIC50 DCSRUN for COUP-TFII (NR2F2)

Protocol: Assay Overview:

The purpose of this assay is to determine whether the hits identified as active in a set of experiments entitled, Luminescence-based cell-based primary high throughput screening assay to identify antagonists of COUP-TFII (NR2F2) (AID 686940), are assay artifacts affecting luciferase activity, cell viability or the transcription/translation machinery in a non-selective manner.

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 9.9 ug of the plasmid encoding VP16 (pAB-Gal4-VP16), 9.9 ug of the p17 mer x4-TK-Luc luciferase reporter plasmid, and 65 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, VP16 inhibition was mimicked by transfecting cells in absence of the VP16 expressing vector (i.e. reporter plasmid only). Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 85.4 uM.

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 VP16 expressed) whereas the rest of the plate was dispensed with VP16-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_Low_Control ) / ( Median_High_Control - Median_Control_Field ) ) * 100

Where:

Low_Control is defined as wells
High_Control is defined as wells containing cells transfected with reporter plamid only (p17 mer x4-TK-Luc)
Test_Compound is defined as well containing cells cotransfected with pAB-Gal4-VP16 and p17 mer x4-TK-Luc in the presence of test compounds


PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

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

List of Reagents:

p17 mer x4-TK-Luc luciferase reporter plasmid (Assay Provider)
pAB-Gal4-VP16 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. The MLSMR was not able to provide all compounds selected for testing in this assay.

External ID: COUPTFII_INH_LUMI_1536_3X%INH CRUN

Protocol: Assay Overview:

The purpose of this cell-based assay is to confirm activity of compounds identified as active in a set oof previous experiments entitiled, "Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of COUP-TFII (NR2F2)" (AID 686940).

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. Compounds are tested in triplicate at a final nominal concentration of 8.5 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 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_Low_Control) / ( Median_High_Control - Median_Low_Field ) ) * 100

Where:
Low_Control is defined as wells
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:

The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the primary screen (AID 686940) 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 100-33, and for inactive compounds 33-0.

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. The MLSMR was not able to provide all compounds selected for testing in this assay.

External ID: PRPC_INH_TRFRET_1536_3X%INH CRUN

Protocol: Assay Overview:

The purpose of this assay is to confirm activity of compounds identified as active in a set of previous experiments entitled, "TRFRET-based cell-based primary high throughput screening assay to identify inhibitors of cell surface Prion Protein (PRPC)" (PubChem AID 720596). 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 triplicate at a final nominal concentration of 13.8 micromolar.

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 wells containing cells and DMSO.

PubChem Activity Outcome and Score:

The average percent inhibition and standard deviation of each compound tested were calculated. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the primary screen (AID 720596) 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 100-16, and for inactive compounds 16-0.

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. The MLSMR was not able to provide all compounds selected for testing in this assay.

External ID: HGDAF12_AG_LUMI_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as agonists of the nuclear receptor daf-12. In this assay, CV-1cells are co-transfected with a GAL4-responsive reporter plasmid (UAS-tk-luc) and expression vector encoding GAL4-hgDAF12. The ability of compounds to increase transcriptional activity is assessed by measuring luciferase expression from the reporter gene plasmid. Compounds were tested in singlicate at a final nominal concentration of 6.8 micromolar.

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 8 ug of the hgDAF12-expressing vector, 20 ug of the MH1000-tk-luc reporter plasmid and 80 uL of transfection reagent. Twenty four hours post transfection, cells were harvested using 5 mL of preheated TrypLE and resuspended at a concentration of 1 million cells per mL in phenol-red free DMEM supplemented as above. Sodium butyrate, a compound shown to increase the luminscent signal in this assay, was used as a positive control.

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) and placed in the incubator for 3 hours. Cells were then treated with 34 nL/well of either test compounds, DMSO (Low Control, final concentration 0.68%) or 2 mM of Sodium Butyrate (High Control). Plates were incubated for 24 hours at 37 C, 5% CO2 and 95%RH and 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 15 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer).

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

%_Activation = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median__Low_Control ) )

Where:

High_Control is defined as wells treated with 2 mM Sodium Butyrate
Low_Control is defined as wells treated with DMSO only.
Test_Compound is defined as wells treated with test compound.

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 activation. Negative % activation values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-12, and for inactive compounds 12-0.

List of Reagents:

MH1000-tk-luc luciferase reporter plasmid (Assay Provider)
hgDAF12 expressing plasmid (Assay Provider)

List of Consumables:

Sodium Butyrate (Sigma, part B5887)
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)
One-Glo (Promega, part E6130)
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 luciferase activity and hence 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.

In absence of a Protein Entry for the DAF-12 protein from H. glycines in NCBI's database, links to the target sequence and origin refer to the closest known orthologue from the prototypic parasitic helminth C.elegans.

External ID: COUPTFII_INH_LUMI_1536_3XIC50 DRUN

Protocol: Assay Overview:

The purpose of this assay is to determine dose response curves for compounds that confirmed inhibitory activity in a set oof previous experiments entitiled, "Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of COUP-TFII (NR2F2)" (AID 686940, 687008).

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. Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 85.4 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 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_Low_Control) / ( Median_High_Control - Median_Low_Control ) ) * 100

Where:

Low_Control is defined as wells
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

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 85.4 uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 85.4 uM.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

The PubChem Activity Score range for active compounds is 100-97, and for inactive compounds 97-0.

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. The MLSMR was not able to provide all compounds selected for testing in this assay.

External ID: TPD2_INH_EPIABS_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as inhibitors of the activity of tyrosyl DNA phosphodiesterase 2 (TDP2). TDP2 is a divalent cation-dependent enzyme that repairs TopII-associated DNA strand breaks. It is hypothesized that inhibitors of TDP2 may serve as useful adjuvants in combination with cancer drugs such as etoposide.

In this biochemical assay, recombinant human TDP2 protein is incubated at 37 degrees Celsius with the T5PNP substrate in the presence of Mg2+-containing assay buffer.T5PNP is a substrate for snake venom phosphodiesterase, as well as a substrate for TDP2. As a substrate for TDP2, T5PNP is used to mimic the TopII-DNA complex. TDP2 cleaves the phosphodiester bond in T5PNP, and the chromogenic p-nitrophenol group is released. As time increases the TDP2 enzyme will increasingly catalyze hydrolysis of the T5PNP substrate, resulting in increased release of p-nitrophenol and detection at 415nM wavelength. Compounds are tested in singlicate at a final nominal concentration of 12.8microM.

Protocol Summary:

Prior to the start of the assay, 2 ul of a solution containing T5PNP subtrate (final concentration 5mM) in assay buffer (50mM Tris-HCl pH7.5, 1mM DTT, 1mM MgCl2, 50mM KCl and 100ug/ml BSA) was dispended into a all wells of a 1536 well plate. Next, 39nL of test compound in DMSO or DMSO alone (1% final concentration) was added to the appropriate wells. The assay was started by dispensing 1 ul of a solution contanting TDP2 enzyme (120nM final concentration) in assay buffer to wells in columns 4-48 and 1ul of assay buffer alone to wells in columnes 1-3. Plates were centrifuged and incubated for 2hrs at 37 degrees Celsius at which time fluorescence intensity was measured (Ex. 405nm and Em. 405nm) using a EnVision microplate reader (Perkin Elmer).

Prior to further calculations, the following formula was used to calculate Epi Absorbance (EPIABS)

EPIABS = -log10( sample / background)

Where:

Sample is defined as the fluorescent intensity of wells containing test compounds or DMSO.
Background is defined as the fluorescent intensity of wells containing buffer and T5PNP only.

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

%_Inhibition = ( EPIABS_Test_Compound - MedianEPIABS_Low_Control ) / ( MedianEPIABS_High_Control - MedianEPIABS_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing test compound, TDP2 and T5PNP.
High_Control is defined as wells containing only Buffer and T5PNP.
Low_Control is defined as wells containing DMSO, TDP2 and T5PNP.

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.

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 PubChem Activity Score range for active compounds is 100-22, and for inactive compounds 22-0.

List of Reagents:

TDP2 (supplied by Assay Provider)
Tris Base(Sigma, 93349)
DTT (Sigma, 43815)
BSA (Sigma, A2153)
MgCl2 (Sigma,M2670)
KCl (Sigma, P9333)
T5PNP (Sigma, T4510)
1536 well plates (Corning 7254)

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: 7017-01_Other_SinglePoint_HTS_Activity

Protocol: Protocol:

Cystic Fibrosis airway epithelial cells (CFBE) have been transiently transfected (electroporation) with vectors containing an Yellow Fluoresencent Protein (YFP) halide-sensitive mutant and the human Cystic Fibrosis Transmembrane conductance Regulator with the deletion of the amino acid phenylalanine 508 (CFTR-F508del) provided by Dr. Jinliang Sui from the Flatley Discovery laboratory (Charlestown, MA).

Day 1 (Cell plating and compound pinning in assay plates)

Prior to perform the luciferase reporter assay experiments, the CFBE cells are thawed and reconstituted in MEM medium (Invitrogen, 11965) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS) (Gibco, 16140), 1X antibiotic (Penicillin/Streptamycin/Glutamine) (Gibco, Ref. no. 10378-016). CFBE cells are then plated in 384-well format cell culture treated solid black wall clear bottom assay plates (Corning, Cat. # 3712) at a density of 6,000 cells/well using the multidrop dispenser (standard cassette)(Thermo Scientific) in a final volume of 50 ul. The assay plates are placed in 5% CO2 incubator where they are incubated for 2h at 37oC. After 2h of incubation, the assay plates are pulled out of the incubator and are placed side by side on a pinning table adjacent to compound plates containing the MLPCN library and a sentinel plate containing 32 wells with the positive control C18. 100 nl of the compounds and the positive control C18 (final concentration 10 uM) are then collected on metal pins from these compound plates and transferred to the assay plate. The pins are washed with DMSO and methanol between each transfer. The assay plates are then moved back to the 5% CO2 incubator and incubated for an additional 24h.

Day 2 (Cell wash, CFTR channel activation, quenching and reading on the Flipr)

The assay plates are coming out of the incubator and washed once with PBS (50 ul) using a Biotek plate washer and 20ul of 20 uM Forskolin (Cayman Chemicals) and 3uM potentiator P3 (final concentration) in DPBS is added to the assay plate for 1h at 37oC. Then, the assay plates are cooled down for 30 minutes at room temperature and 25 ul of YFP quencher sodium iodide (72.5 mM final concentration) in DPBS is added and the fluorescence is measured every second for one minute on the Flipr tetra (Molecular Devices).

DPBS (Dulbecco's Phosphate buffered saline)
2.7 mM KCL
8.1 mM Na2PO4
1.5 mM KH2PO4
0.7 mM CaCl2*2H2O
1.1 mM MgCl2

DPBS NaCl
137 mM NaCl

DPBS NaI
145 mM NaI

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC) 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 8.

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: PLCG1_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-G1. In this assay, PLC-G1 isozyme is incubated with test compounds and fluorogenic reporter WH-15. As designed, test compounds that act as PLC-G1 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-G1 at a final concentration of 5pg/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 PLCG1 in the presence of test compound and WH15 fluoreogenic reporter.
High_Control is defined as wells containing PLCG1, 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 100-11, for inactive 11-0.

List of Reagents:

PLCG1 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: FIBRIN-TN7_INH_FP_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that bind to Fibrin-DD(E), a protein substrate prepared from human-derived polymerized fibrin clots. In this assay, Fibrin-DD(E) protein is incubated with test compounds for a defined period, followed by addition of fluorescent probe fluorescein Tn7 (FITC-Tn7). The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. As designed, test compounds that bind to Fibrin-DD(E) will prevent Fibrin-DD(E)-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds will be tested in singlicate at a final nominal concentration of 8.3 uM.

Protocol Summary:
Displacement assay starts by the addition of Fibrin-DD(E) (final concentration 2uM) in assay buffer (50mM Tris base, 100 mM NaCl, 2 mM CaCl2, and 0.01% Triton X-100, pH 7.8) to all wells, followed by the addition of equal volume of inhibitor peptide Aha-Tn7 (final concentration 10uM) to high control wells and equal volume of assay buffer to sample wells and low control wells. Compounds in DMSO are transferred to plate and incubated for a period of 10 min, at which time TRITC-TN7 probe is added to all wells (final concentration of 0.1uM). After 3 hrs at room temperature fluorescence polarization is measured using Perkin Elmer EnVisionwith FITC filter set.


%_Inhibition = ( FP_Test_Compound - MedianFP_Sample_Field ) / ( MedianFP_High_Control - MedianFP_Sample_Field ) * 100

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing Aha-Tn7 peptide.
Low_Control is defined as wells containing DMSO.
Sample_Field is defined as 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 100-15, for inactive 15-0.

List of Reagents:
Fibrin-DD(E ) (Supplied by Assay Provider)
FITC-Tn7 peptide (Supplied by Assay Provider)
Aha-Tn7 peptide (Supplied by Assay Provider)
TRIZMA(R) Base BioXtra >99% (Sigma-Aldrich, part T6791)
Tritontrade mark X-100 BioXtra (Sigma-Aldrich, part T9284)
Sodium Chloride BioXtra >99.5% (Sigma-Adrich, part S7653)
Calcium Chloride Dihydrate BioXtra >99% (Sigma-Aldrich, part C5080)
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: FIBRIN-TN6_INH_FP_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that bind to Fibrin proteolytic fragment-DD(E), a protein substrate prepared from human-derived polymerized fibrin clots. In this assay, Fibrin-DD(E) protein is incubated with test compounds for a defined period, followed by addition of the fluorescent probe tetramethylrhodamine Tn6 (TRITC-Tn6). The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value. As designed, test compounds that bind to Fibrin-DD(E) will prevent Fibrin-DD(E)-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds will be tested in singlicate at a final nominal concentration of 8.3 uM.

Protocol Summary:
Displacement assay starts by the addition of Fibrin - DD(E) (final concentration 2uM) in assay buffer (50mM Tris base, 100 mM NaCl, 2 mM CaCl2, and 0.01% Triton X-100, pH 7.8) to all wells, followed by the addition of equal volume of inhibitor peptide EP-2104R (final concentration 10uM) to high control wells and equal volume of assay buffer to sample wells and low control wells. Compounds in DMSO are transferred to plate and incubated for a period of 10 min, at which time TRITC-TN6 probe is added to all wells (final concentration of 0.1uM). After 3 hrs incubation at room temperature fluorescence polarization is measured using Perkin Elmer EnVision with TRITC filter set.



%_Inhibition = ( FP_Test_Compound - MedianFP_Sample_Field ) / ( MedianFP_High_Control - MedianFP_Sample_Field ) * 100

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing EP-2104R peptide.
Low_Control is defined as wells containing DMSO.
Sample_Field is defined as 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 100-1, for inactive 1-0.

List of Reagents:

Fibrin-DD(E ) (Supplied by Assay Provider)
TRITC-Tn6 peptide (Supplied by Assay Provider)
EP-2104R peptide (Supplied by Assay Provider)
TRIZMA(R) Base BioXtra >99% (Sigma-Aldrich, part T6791)
Tritontrade mark X-100 BioXtra (Sigma-Aldrich, part T9284)
Sodium Chloride BioXtra >99.5% (Sigma-Adrich, part S7653)
Calcium Chloride Dihydrate BioXtra >99% (Sigma-Aldrich, part C5080)
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: BRM_ACT_LUMI_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as activators (re-activators) of BRM function. This assay relies on the fact that BRM assists the glucocorticoid receptor (GR) in activating the MMTV promoter, which is used to drive expression of a luciferase reporter gene (7). Compounds active in this assay will restore BRM function, allowing the SWI/SNF-dependent GR to induce the MMTV promoter, leading to increased luciferase expression and well luminescence. MG213 cells are incubated in the presence of 0.1 uM Dexamethasone for 24 hours prior to be plated in 1536-well plates at 600 cells/well in 5 ul media with Dexamethasone using Kalypsys dispenser. The cells are immediately pinned with 45 nL of compound, control or DMSO, spun down, and incubated overnight at 37 C. Test compounds compound are delivered to the plates containing the MG213 cells and dexamethasone using pintool. Chembridge 5306793 is included as a positive control on each plate. OneGlo luciferase reagent is used according to the manufacturer's directions. Luminescence is measured using the Viewlux plate reader. Compounds are tested in singlicate at a final nominal concentration of 9.1 uM. Note that the cell line used for this assay has been transduced with the E1A gene, which produces the viral oncoprotein, E1A. This protein sequesters Rb (and Rb2) protein thus preventing cellular growth inhibition. At lower densities, these newly modified cells have a more linear growth rate and do not lag when plated at low density. At higher densities (which typically impacts normal cell growth via contact inhibition), these cells maintain their growth rate allowing these cells to grow to higher densities. This modified cell line allows for a greater experimental range for this assay and improves its performance (consistency) as compared to the parental MG213 cells.

Protocol Summary:

The MG132 cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of RPMI -1640 supplemented with 5% v/v certified fetal bovine serum, 500 ug/mL Geneticin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).

Prior to the start of the assay 600 cells in a 5 ul volume of assay media (growth media as above except without geneticin and with 2.5% FBS and 0.1uM Dexamethasone) were dispensed into each well of 1536-well tissue culture-treated microtiter plates. The assay was started immediately by dispensing 45 nL of test compound in DMSO (0.69 % final DMSO concentration), DMSO alone, or CB5306793 to the appropriate wells. Next, the plates were incubated for 24 hours at 37 C (5% CO2, 95% RH). The assay was stopped by dispensing 5 ul of One Glo luciferase substrate to each well, followed by incubation at room temperature for 15 minutes. Well luminescence was measured on the ViewLux plate reader.

The percent activation for each compound was calculated as follows:

%Activation = ( 1 - ( ( Test_Compound - Median_High_Control ) / ( Median_Low_Control - Median_High_Control ) ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing CB5306793.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally activating compounds in the Primary Screen. Two values were calculated for each assay plate: (1) the average percent activation of test compound 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 % activation than that particular plate's cutoff parameter was declared active. The reported PubChem Activity Score has been normalized to 100% of the highest observed primary activation value. Negative % activation values are reported as activity score zero.

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

The PubChem Activity Score range for active compounds is 100-6, and for inactive compounds 6-0.

List of Reagents:

MG132 cell line (provided by Assay Provider)
RPMI-1640 medium (Invitrogen, 11875-119
Geneticin (Invitrogen, part 10131-035)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16000-044)
One Glo Assay Kit (Promega, part E6130/QTE30675 )
CB5306793, Chembridge
T-175 tissue culture flasks (Corning, part 431080)
1536-well plates (Corning, 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. 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 non-specifically modulate luciferase activity, and compounds that quench or emit luminescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: MBD2-CPGDNA_INH_TRFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that inhibit the binding of cloned recombinant 6-His-tagged DNA-binding domain of MBD2 (MBD2-DBD) to a FAM-labeled 14 bp hairpin oligonucleotide containing a symmetrically-methylated CpG, whose interaction is monitored using Tb labeled anti-his antibody. Pre-incubation of the MBD2 protein, FAM labeled methylated CpG oligo and Tb-labeled anti-His antibody leads to formation of a stable complex. Anti-his-complexed lanthanide (Tb) attaches to the anti-his-binding peptide tag on MBD2-DBD protein. When terbium donor is excited, Tb transfers energy to a fluorescein tag on the methylated oligo, now complexed with MBD2-DBD, resulting in an increase in TR-FRET. An inhibitor of the MBD2-DBD:FAM methylated CpG interaction will inhibit binding between MBD2 and FAM methylated 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 uM.

Protocol Summary:

The assay was started by dispensing 5 uL 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 25 nM of MBD2 protein, 30 nM of FAM labeled Unmethylated CpG DNA oligo and 5 nM of Terbium labeled anti-his antibody into columns 1 thru columns 3 of 1536 microtiter plates. Next, 5 uL of Experimental Mix containing 25 nM of MBD2 protein, 30 nM FAM labeled Methylated CpG DNA oligo and 5 nM Terbium labeled anti-his antibody in assay buffer were dispensed into columns 4 thru 48. Then, the plates were centrifuged and pinned with 22 nL of test compound in DMSO or DMSO alone (0.44% final concentration). The plates were incubated for 30 minutes at 25 C 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 Control Mix 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 100-11, and for inactive compounds 11-0.

List of Reagents:

MBD2-MBD protein (supplied by Assay Provider)
FAM labeled Methylated CpG DNA Oligo (IDT-custom order)
FAM labeled Unmethylated 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.