External ID: CHEMBL2216514

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2216515

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: OPRM1-OPRD1_AG_LUMI_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that activate heterodimer formation between the mu (OPRM1) and delta (OPRD1) opioid receptors, resulting in membrane recruitment of beta-arrestin. The assay monitors GPCR-beta-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). This assay employs U2OS cells which express OPRD1, OPRM1 fused to a beta-gal peptide fragment (enzyme donor), and beta-arrestin fused to the complementary beta-gal fragment (enzyme acceptor). Cells are incubated with test compound, followed by measurement of well luminescence. As designed, compounds that induce formation of OPRD1 homodimers or OPRM1-OPRD1 (Mu-Delta) heterodimers will cause beta-arrestin recruitment, resulting in reconstitution of the beta-gal holoenzyme. The reconstituted holoenzyme can then catalyze the hydrolysis of a substrate which yields a chemiluminescent signal, resulting in increased well luminescence. Deltorphin B will be used as the high (100% RLU) control for agonists, and wells containing cells treated with DMSO will be used as the low (0% RLU) control. Compounds were tested in singlicate at a final nominal concentration of 9.3 uM.

Protocol Summary:

The U2OS-OPRM1-OPRD1 (Mu-Delta) cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of a 1:1 mixture of Ham's F-12 Nutrient Media (F-12) and Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% v/v heat-inactivated certified fetal bovine serum, 25 mM HEPES, 250 ug/mL Geneticin, 250 ug/mL Hygromycin B, 0.25 ug/mL Puromycin and 1X antibiotic mix (penicillin, streptomycin, and neomycin).

The day before the assay 1000 cells in 3 uL of cell plating media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 23 hours. Next, 28 nL of test compound in DMSO, Deltorphin B (0.9 uM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. The plates were then incubated for 3 hours at 37 C, 5% CO2, and 95 % RH. The assay was started by adding 2 uL of PathHuntertrade mark reagent (prepared according to the manufacturer's protocol); followed by 1 hour incubation at room temperature. Then, Well Luminescence was read on the ViewLux plate reader

The percent activation for each compound was calculated as follows:

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

Where:

High_Control is defined as wells containing cells, Deltorphin B and DMSO.
Test_Compound is defined as wells containing cells, test compounds and DMSO.
Low_Control is defined as wells containing cells and DMSO.

A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation 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 % activation than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

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 100-2, and for inactive compounds 2-0.

List of Reagents:

PathHuntertrade mark B-arrestin recruitment assay, containing the U2OS OPRM1 OPRD1 Beta-arrestin cell line; PathHunter Detection Kit (DiscoveRx, part, 93-0558C3)
Ham's F-12 media (Invitrogen, part 11765-054)
DMEM media (Invitrogen, part 11995-073)
Detachin (Genlantis, part T100100)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082-147)
Puromycin (Invitrogen, part A11138-02)
Hygromycin B (Invitrogen, part 10687-010)
Geneticin (Invitrogen, part 10131-027)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Nunc, part 159910)
Agonist: Deltorphin B (Anaspec, part 62683)
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 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: CHEMBL2216512

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

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: CHEMBL2216513

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2209386

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: ERK5 transcriptional activity-HTS

Protocol: Stable cells plated on a 384-well plate (2500 cells/well) were treated with test compounds at the concentration of 5 muM for 18 hrs. The level of luciferase activity was assayed using a
Luciferase kit (Promega corporation, Madison, WI) and a series of positive and negative control compounds were used as references.

Comment:

External ID: CHRM1_AG_FLUO8_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as agonists of the human M1 muscarinic receptor (CHRM1; M1). In this assay, CHO-K1 cells stably expressing human M1 are loaded, intracellularly with the calcium indicator dye, Fluo-8, followed by treatment with agonist control or test compounds. As designed, compounds that act as CHRM1 agonists will increase intracellular calcium mobilization, resulting in increased relative fluorescence of the indicator dye and well fluorescence. Compounds are tested in singlicate at a final nominal concentration of 3 uM.

Protocol Summary:

The CHO-hM1 cell line was routinely cultured in T-175 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 20 mM HEPES, 50 ug/mL Geneticin, and 1X antibiotic mix (penicillin and streptomycin).

The day before the assay 3000 cells in 3 uL of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 17-24 hours. Next, 2 uL of the fluorogenic Fluo-8 intracellular calcium indicator mixture (prepared according to the manufacturer's protocol) was added to each well. Plates were then incubated for 1 hour at 37 C, 5% CO2, and 95 % RH, followed by 30 minute incubation at room temperature. Then, 15 nL of test compound in DMSO were dispensed to appropriate wells. The assay was started by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay. A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:

Ratio = I_Max / I_Min

Where:

I_Max represents the maximum measured fluorescence emission intensity over the 140 second read.
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.

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

%_Activation = ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_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 Acetylcholine (EC100) and DMSO.

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 the entire run, i.e. any compound that exhibited greater % activation than the entire screen's 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 100-1, and for inactive compounds 1-0.

List of Reagents:

Cell line: Chinese Hamster Ovary (CHO) cells containing hM1 receptor; (Conn Lab)
Calcium sensitive dye: Fluo-8 No Wash Calcium Assay Kit; (AAT Bioquest, part 36316)
Growth media: Ham's F-12; 10% FBS, 20mM HEPES, 50 ug/mL G418
Assay media: Ham's F-12, 10% FBS, 20 mM HEPES
Assay plates: Aurora black/clear 1536well FLIPR plate; (Aurora, part 00019326)
Probenecid: 250 mM (pH 8.0); (Sigma P8761)
Agonist: Acetylcholine (50 mM stock in water); Sigma A9187

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: CHEMBL2216516

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2209385

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2216506

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2216504

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

External ID: 2147-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: Protocol:
The FadD28 is purified through Ni-NTA affinity column due to a 6x Histidine tag. Purified protein is stored at -80 degrees C (D.J. Wilson, and C.C. Aldrich. Anal. Biochem. 404 (2010) 56-63)
I. Solution preparation (For a run of 121 plates):
1) Prepare 700mL of 200nM compound 11 (TAMRA labeled substrate) in FP buffer. Take 1.4mL of 100uM compound 11 in 100% DMSO in 700mL FP buffer
2) Prepare 50mL compound24 (non-labeled substrate) in FP buffer. Take 200uL of 10mM compound 24 in 100% DMSO in 50mL FP buffer
3) Prepare 350mL 4uM FadD28 in FP buffer. Take 1.746mL of 802uM FadD28 in 350mL FP buffer
II. Setup reagents on automation instrument
1) Add the solutions of compound24, FadD28 and FP buffer to the bottles of BioRaptr (Beckman) based on the dispense table
2) Add compound 11 solution to the bottle of combi NL (Thermo Scientific)
3) Add 1536-well assay ready plates (ARPs) to incubator
III. Run automation protocol
1) Dispense 3uL/well of the solutions from BioRaptr based on the dispense table to 1536-well assay ready plates (ARPs) (Aurora, Cat#: 00019180BX)(Positive control wells receive 1.5uL/well of compound24 and 1.5uL/well of FadD28 solution; all the other wells receive 1.5uL/well of FP buffer and 1.5uL/well of FadD28 solution)
2) Incubate the plates for 10mins at 25 degrees C
3) Dispense 3uL/well of 200nM compound11 solution to the plates by Combi NL
4) Incubate the plates for 30mins at 25 degrees C
5) Read the plates on ViewLux (PerkinElmer) with excitation wavelength of 525nm, emission wavelength of 598 nm and dichroic mirror of 550nm
Final concentration: 100nM compound11, 1uM FadD28, 10uM compound24 (positive control), compound concentration for primary screen: 12.5uM
FP buffer: 30mM Tris-HCl, pH7.5, 1mM MgCl2, 1mM DTT, 0.0025% Igepal CA630

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 Minus Inhibitors' 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 median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the maximum of the normalized 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

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

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: CHEMBL2216509

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2209398

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2209399

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2209396

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2209397

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: 2125-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: Protocol:
PRC2 activity was measured using Dissociation-Enhanced Lanthanide Fluoro-ImmunoAssays (DELFIA) performed on 384-well, white, streptavidin-coated plates (PerkinElmer). In short, PRC2 was diluted to 30 ng and 3 ng per well in 20 uL 2X Enzyme Buffer (50mM Tris HCl pH 8.5, 10 mM DTT, 5 mM MgCl). Compounds were pinned at 100 nL per well and reactions were initiated with 20 uL of a 5 uM SAM (NEB) solution containing 600 nM H3[21-44]-GK-biotin (AnaSpec; PRC2).

Plates were incubated at room temperature for 1 hr and then washed three times with 100 uL of Wash Buffer (50mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween 20, 0.2% BSA). 50 ul of Fluoroimmunoassay (FI) Buffer (50 mM Tris HCl pH 7.8, 150 mM NaCl, 0.05% Tween 40, 25 iM DTPA, 0.2% BSA, 0.05% BGG)containing a 1:4000 dilution of anti-H3K27me2 rabbit IgG (Cell Signaling, #9728) with 691 ng/mL Eu-N1-anti-rabbit IgG (PerkinElmer)was added to each well for PRC2 reactions.

Following 1 hr incubation at room temperature, the plates were washed three times with Wash Buffer and 50 uL of Enhancement Solution (PerkinElmer) was added to each well. Plates were incubated for 30 min at room temperature and time-resolved fluorescence (TRF) was measured on Wallac Envision 2104 Multilabel Reader (400 us window, 400 us delay, 320 excitation, 615 emission).

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 Minus Inhibitors' 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 median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized 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 50.

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 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

External ID: CHEMBL2209401

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL948204

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2008
Volume: 16
Issue: 5
First Page: 2665
Last Page: 2675
DOI: 10.1016/j.bmc.2007.11.038

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2209390

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: ABHD4_INH_FP_1536_3X%INH CRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to confirm activity of compounds identified as active in a previous set of experiments entitled, "Fluorescence polarization-based biochemical high throughput primary assay to identify inhibitors of alpha/beta hydrolase domain containing 4 (ABHD4)" (AID 720543). 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 triplicate 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 wells containing ABHD4 wild type, DMSO and FP-Rh.

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 720543) 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 100-8, for inactive 8-0.

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

External ID: CHEMBL948205

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2008
Volume: 16
Issue: 5
First Page: 2665
Last Page: 2675
DOI: 10.1016/j.bmc.2007.11.038

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2209388

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHRM1_PAM_FLUO8_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as positive allosteric modulators (PAMs) and increase activity of the human M1 muscarinic receptor (CHRM1; M1) in cells pre-treated with a known agonist. In this assay, CHO-K1 cells stably expressing human M1 are loaded with the Fluo-8 calcium indicator dye, followed by addition of test compounds and subsequent treatment with the activator acetylcholine at a concentration that results in 20% activation (EC20). As designed, compounds that act as CHRM1 PAMs will increase calcium mobilization, resulting in increased intracellular calcium and relative fluorescence of the indicator dye beyond that of the EC20 of acetylcholine. Compounds are tested in singlicate at a final nominal concentration of 3 micromolar.

Protocol Summary:

The CHO-hM1 cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Ham's F-12 Nutrient Media (F-12) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 20 mM HEPES, 50 micrograms/mL Geneticin, and 1X antibiotic mix (penicillin and streptomycin).

The day before the assay 3000 cells in 3 microliters of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 degrees C, 5% CO2, and 95 % RH for 17-24 hours. Next, 2 microliters of the fluorogenic Fluo-8 intracellular calcium indicator mixture (prepared according to the manufacturer's protocol) was added to each well. Plates were then incubated for 1 hour at 37 degrees C, 5% CO2, and 95 % RH, followed by 30 minute incubation at room temperature. Then, 15 nL of test compound in DMSO were transferred to appropriate wells. The assay was started by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices) prior to all wells being treated with an EC20 concentration of acetylcholine. Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay. A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:

Ratio = I_Max / I_Min

Where:

I_Max represents the maximum measured fluorescence emission intensity over the 140 second read and;
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.

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

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

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing Acetylcholine at EC20 and DMSO.
High_Control is defined as wells containing Acetylcholine (EC100) and DMSO.

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 on an individual plate basis, i.e. any compound that exhibited greater % activation than the plate based cutoff parameter was declared active.

PubChem Activity Outcome and Score:

The inactive compounds of this assay have an activity score range of 0 to 78 and the active compounds have an activity score range of 50 to 100.

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

List of Reagents:

Cell line: Chinese Hamster Ovary (CHO) cells containing hM1 receptor; (Conn Lab)
Calcium sensitive dye: Fluo-8 No Wash Calcium Assay Kit; (AAT Bioquest, part 36316)
Growth media: Ham's F-12; 10% FBS, 20mM HEPES, 50?g/mL G418
Assay media: Ham's F-12, 10% FBS, 20 mM HEPES
Assay plates: Aurora black/clear 1536well FLIPR plate; (Aurora, part 00019326)
Probenecid: 250mM (pH 8.0); (Sigma P8761)
Potentiator: Acetylcholine (50mM stock in water); Sigma A9187

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: CHEMBL2209395

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 18
First Page: 5537
Last Page: 5549
DOI: 10.1016/j.bmc.2012.07.043

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: epac1-activator-v

Protocol: Briefly, three uL of reagents (100 nM EPAC1, 250 nM RAP1B-BODIPY-GDP, 50 uM GDP) were dispensed into a 1536-well Greiner black solid-bottom medium binding assay plate. Controls and test compounds (23 nL) were transferred to the plate via a Kalypsys pin tool equipped with a 1536-pin array. The plates were centrifuged at 1,000 rpm for 15 seconds followed by 5 minute incubation at room temperature. The assay plates were read at 5 minute intervals for 30 minutes in the ViewLux plate reader using 480nm excitation and 540nm emission filters. The results were normalized to the agonist positive control of 6.5 mM cAMP.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods.

Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, with a ratio activity curve class of 4, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. For a ratio activity curve class = 1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 1.2 || ratio.curve_class == 2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds also have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

External ID: SBCCG-A1015-NR3A-Primary-Assay

Protocol: A.#Brief Description of the Assay
This assay attempts to identify small molecule compound binding to NR3A LBD using differential scanning fluorimetry assay. The assay is performed format using Applied Biosystems 384-well plates (cat #4309849) on ViiA7 qPCR instrument (Thermo-Fisher Scientific).

B.#Assay reagent components:
1.#Assay buffer: 20 mM HEPES, pH 7.5, 200 mM NaCl
2.#NR3A LBD working solution in the assay buffer
3.#NR3A LBD with glycine working solution in the assay buffer
4.#Sypro Orange working solution in the assay buffer
5.#Compounds in 100% DMSO

C.#Step-by-step protocol:
1.#Reagent dispenses
a.#Add compound aliquots to the wells in columns 3-24
b.#Add DMSO aliquots to the wells in columns 1-2
c.#Dispense 5 uL of NR3A LBD working solution into columns 2-24
d.#Dispense 5 uL of NR3A LBD with glycine working solution to column 1
e.#Dispense 5 uL of Sypro Orange with glycine solution to columns 1-24
2.#Perform assay by ramping temperature and measuring concomitant changes in fluorescence
3.#Determine Tm corresponding to the maximum of the first derivative of fluorescence
D.#Final concentration of reagents in the assay wells
1.#1.25 uM NR3A LBD (all wells)
2.#x5 Sypro Orange (all wells)
3.#25 uM tested compounds (wells in columns 3-24)
4.#0.25% DMSO (all wells)
E.#Plate Map:
1.#Positive (high Tm) control in column 1
2.#Negative (low Tm) control in column 2.
3.#Test wells in columns 3-24.

Comment: %Activity = (Melting temperature of compound well - Average melting temperature of negative control well)/(Melting temperature of positive control wells - Melting temperature of negative control wells)*100%.

Compounds that demonstrated %Activity >= 10% at 25 uM concentration are defined as actives in this assay.

Activity Scoring
The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is active, then the assigned score is 20

External ID: epac1-inhibitor-v

Protocol: Briefly, three uL of reagents (100 nM EPAC1, 250 nM RAP1B-BODIPY-GDP, 50 uM GDP) were dispensed into a 1536-well Greiner black solid-bottom medium binding assay plate. Controls and test compounds (23 nL) were transferred to the plate via a Kalypsys pin tool equipped with a 1536-pin array. The plates were centrifuged at 1,000 rpm for 15 seconds followed by 5 minute incubation at room temperature. The assay plates were read at 5 minute intervals for 30 minutes in the ViewLux plate reader using 480nm excitation and 540nm emission filters. The results were normalized to the agonist positive control ATA and DMSO.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods.

Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with a ratio activity curve class of 4, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. For a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

External ID: CHEMBL947191

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2008
Volume: 16
Issue: 5
First Page: 2665
Last Page: 2675
DOI: 10.1016/j.bmc.2007.11.038

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL947192

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2008
Volume: 16
Issue: 5
First Page: 2665
Last Page: 2675
DOI: 10.1016/j.bmc.2007.11.038

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

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: 2084-01_Activator_SinglePoint_HTS_Activity

Protocol: MITF Primary Screening Protocol
(TRPM-1 Promoter//Luciferase reporter assay)

Day 1, plate cells 2000 per well in 30 uL media (phenol red free DMEM/10% iFBS/Pen/Strep/L-Glutamine)

Day 2, pin 100 nL into 30 uL assay volume in white, opaque Corning 8867 barcoded 384 well plates. (will also require sentinel pinning with the positive control, parthenolide)
Incubate 24 hours at 37 degrees C in Liconic incubator

Day 3, add 20 uL 100% Promega Steady glo with Thermo Combi fluid transfer apparatus.
Shake 15 seconds on "big bear" plate shaker.
Incubate at RT for 5 minutes.
Read on Perkin-Elmer Envision with US LUM settings for 0.5 sec per well

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

EXPECTED OUTCOME: Active compounds result in increasing readout signal.
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' 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 median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) 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 80.

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: PolI100

Protocol: Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol iota in columns 1, 2, and 5-48) will be dispensed into 1,536-well black solid-bottomed plate. Compounds (23 nL) will be transferred via Kalypsys pin tool equipped with 1536-pin array. The plates will then be incubated for 15 min at room temperature, and 1 muL substrate (50 nM final concentration) will be added to start the reaction and kinetically read twice at 0 min and 10 min on the Viewlux reader.

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: CHEMBL947193

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2008
Volume: 16
Issue: 5
First Page: 2665
Last Page: 2675
DOI: 10.1016/j.bmc.2007.11.038

Target ChEMBL ID: CHEMBL612794
ChEMBL Target Name: U-937
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: epac2-activator-v2

Protocol: Briefly, three uL of reagents (100 nM EPAC2, 250 nM RAP1B-BODIPY-GDP, 50 uM GDP) were dispensed into a 1536-well Greiner black solid-bottom medium binding assay plate. Controls and test compounds (23 nL) were transferred to the plate via a Kalypsys pin tool equipped with a 1536-pin array. The plates were centrifuged at 1,000 rpm for 15 seconds followed by 5 minute incubation at room temperature. The assay plates were read at 5 minute intervals for 30 minutes in the ViewLux plate reader using 480nm excitation and 540nm emission filters. The results were normalized to the agonist positive control of 6.5 mM cAMP.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods.

Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. For this assay, apparent activators are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, with a ratio activity curve class of 4, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. For a ratio activity curve class = 1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == 1.2 || ratio.curve_class == 2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds also have PUBCHEM_ACTIVITY_SCORE between 1 and 39.

External ID: GDH-TPI_INH_ABS_1536_1X%INH CSRUN

Protocol: Assay Overview:

The purpose of this biochemical counterscreen is to determine whether compounds act as absorbance assay artifacts or are non-selective. This assay also serves as a counterscreen for a set of ongoing high throughput primary experiments entitled, "Absorbance-based biochemical primary high throughput screening assay to identify inhibitors of the aldolase of M. tuberculosis."

This counterscreen is similar in format to the aforementioned assay with the only two following differences: (i) the fructose-1,6-bisphosphate substrate is replaced with glyceraldehyde 3 phosphate, a product of its conversion by FBA and (ii) no (FBA) is used. The counterscreen hence recapitulates the two steps involved in the monitoring of FBA activity through the conversion of FB into the triose product glyceraldehyde 3 phosphate (G3P), which would be converted to dihydroxyacetone phosphate (DHAP) by the helper enzyme triose phosphate isomerase (TPI). A second helper enzyme, glycerophosphate dehydrogenase (GDH), converts the dihydroxyacetone phosphate to glycerol-3-phosphate with the concomitant oxidation of NADH to NAD, which is monitored by measuring the absorbance at 340 nm. In this new assay format, the A340 is independent of FBA activity, hence compounds that reduce absorbance at 340 nm are either absorbance artifacts or helper enzyme inhibitors that will not be pursued. Compounds are tested in singlicate at a final nominal concentration of 4.78 uM.

Protocol Summary:

Prior to the start of the assay, 5 uL /well of Buffer A (50 mM HEPES, 0.01% Triton X-100, 10% Glycerol, pH8.0) supplemented with 400 nM ZnCl2,240 uM NADH and the helper enzymes GDH-TPI (4 U/mL) was dispensed into all wells of a 1536-well plate except the "No enzyme" wells that contained the same supplemented buffer but no GDH-TPI enzymes. Next, 48 nL of test compounds were then delivered in each well using a PinTool. The assay was then initiated by dispensing 5 uL of Buffer A supplemented with 240 uM of the substrate glyceraldehyde-3-phosphate (G3P). Plates were incubated at room temperature for 20 minutes before A340 was measured using the EnVision plate reader (Perkin Elmer).

The percent inhibition for each compound was calculated as follows:

%Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

Test_Compound is defined as wells treated with test compounds.
Low_Control is defined as wells treated with DMSO.
High_Control is defined as wells with no GDH-TPI enzyme.

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 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 the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

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

The PubChem Activity Score range for active compounds is 100-13, and for inactive compounds 13-0.

List of Reagents:

ZnCl2 (Fisher Scientific, part Z33-500)
NADH (EMD Biosciences, part 481913)
GDH-TPI (Sigma, part G1881)
HEPES (EMD Biosciences, part EM-5310)
Triton X-100 (Sigma, part T8787)
Glycerol (Fisher, part AC327255000)
Glyceraldehyde-3-phosphate (Sigma, part D7137)
1536-well plates (Aurora, part 1091-11020-S)

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, and compounds that quench or emit absorbance 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: PolE100

Protocol: Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol eta in columns 1, 2, and 5-48) were dispensed into a 1,536-well black solid-bottomed plate. Compounds (23 nL) were transferred via Kalypsys pin tool equipped with 1536-pin array. The plates were then incubated for 15 min at room temperature, and 1 muL substrate (50 nM final concentration) was added to start the reaction and kinetically read twice at 0 min and 10 min on the Viewlux reader

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: epac2-inhibitor-v2

Protocol: Briefly, three uL of reagents (100 nM EPAC2, 250 nM RAP1B-BODIPY-GDP, 50 uM GDP) were dispensed into a 1536-well Greiner black solid-bottom medium binding assay plate. Controls and test compounds (23 nL) were transferred to the plate via a Kalypsys pin tool equipped with a 1536-pin array. The plates were centrifuged at 1,000 rpm for 15 seconds followed by 5 minute incubation at room temperature. The assay plates were read at 5 minute intervals for 30 minutes in the ViewLux plate reader using 480nm excitation and 540nm emission filters. The results were normalized to the agonist positive control of 6.5 mM cAMP.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods.

Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, with a ratio activity curve class of 4, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. For a ratio activity curve class = -1.1, score = 80+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 && abs(ratio.max_response) > 6*10, score = 60+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -2.1 || ( ratio.curve_class==-2.2 && abs(ratio.max_response) > 6*10), score = 40+abs((log_ac50+4.5)*inf_activity/20). For ratio.curve_class == -1.2 || ratio.curve_class == -2.2, score = 20+abs((log_ac50+4.5)*inf_activity/20). Inconclusive compounds, with a donor curve class other than 4, have PUBCHEM_ACTIVITY_SCORE of 10. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39.