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: 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: 2134-01_Inhibitor_SinglePoint_HTS_Activity_Set2

Protocol:
1. Clone20-Shn3FFL cells (Lot#1-810) are cultured and expanded in OB media
to obtain sufficient cell numbers to plate across the ~2,100 384-well plates required
to screen in duplicate the 330,000 compounds in the MLSMR collection.
2. Cells are plated at a concentration of 3000 cells per well in 30ul of OB media using a Combi Multidrop (Thermo)
in white opaque 384-well plates (Corning 3570/8867BC).
3. Once plated, cells are incubated overnight at 37 degrees C 5% CO2 95% humidity prior to the addition of
compounds.
4. Compounds are then be added to plates at a volume of 100nl of compound using a steel pin tool (V&P Scientific).
5. Following the addition of compounds, cells will be incubated for an additional 24
hours at 37 degrees C 5%CO2 95% humidity.
6. After 24 hour incubation period, cells and luciferase reagents are allowed to
equilibrate to room temperature for 10 minutes.
7. For firefly luciferase assay, 20ul of Cell Titer Glo luciferase (Promega) is added
to each well with a Combi multidrop and then plates are incubated for 15 minutes at room temperature
on orbital shaker.
8. Plates are read on an Envision multi-mode plate reader (Perkin Elmer) to
obtain luminescence readings (0.1 s/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 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: 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 75.

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

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: CGM data for Cell Systems paper Dec 2015

Protocol: Yeast strains and Media:
All S. cerevisiae deletion strains were obtained from the Euroscarf deletion collection. The wild type parental strain for this collection is BY 4741 MATa his31 leu20 met150 ura30. An isogenic pdr1pdr3 strain (MT2481) was created using PDR1::nat and PDR::URA3 deletion cassettes. All strains were grown and screened in synthetic complete (SC) medium with 2% glucose.

Handling:
Strains were seeded at 50,000 cells per well in a volume of 100 L in 96 well plates followed by addition of 2 L of 1 mM compound stock for final concentration of 20 M. Screens were conducted in technical duplicate using a Biomek FX liquid handling workstation with an integrated stacker carousel. DMSO solvent only controls and 10 uM cycloheximide positive controls were seeded in columns 1 and 12 of each assay plate. Plates were incubated at 30 C without shaking for approximately 18 h or until culture saturation was achieved for the solvent controls. Cultures were resuspended by shaking on the robotic platform prior to reading OD600 values on either Tecan M1000 or Tecan Sunrise plate readers.

Analysis procedure:
All data was subjected to the following workflow:
1. Consistent spatial effects on growth across plates were corrected by Lowess regression using an empirically estimated sliding window of 1/3 and normalized based on plate median.
2. If more than 30% of compounds were active within a plate, data was normalized to DMSO controls.
3. Median normalization was applied to all plates and experiments.
4. Z-factors for growth inhibition were calculated using the median and the interquartile range (IQR) by fitting a normal distribution with N(1,IQR) to the experimental data. In addition, we calculated the Z-factor, percent inhibition and normalized OD values for manual validation.
5. Data points with high variation between replicates (> 3 MAD) were removed as inconsistent outliers.

BioAssay submission file header:

Pubchem_ext_datasource_regid: compound identifier used in on chemgrid.org/cgm
Orf yeast gene deletion (open reading frame)
Sym# yeast gene deletion gene symbol
Raw OD read 1 first replicate
Raw OD read 2 second replicate
Normalized OD average normalized read based on workflow described above
Z_score Z-Score calculated based on kernel density distribution from normalized average reads per screen
P_value p-value calculated based on kernel density distribution calculated from average reads per screen
Non replicate test for non-replicates between first and second replicate
Pubchem_activity_outcome pubchem activity outcome
Cryptagen if compound identifier fulfilled cryptagen rule of activity in at least 4 and not more than 2/3rds of screens per library
Bioactivity sensitive if compound decreases fitness or resistant if compound increases fitness compared to negative control

Comment:

External ID: CHEMBL2318170

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: Bioorg. Med. Chem.
Year: 2013
Volume: 21
Issue: 1
First Page: 178
Last Page: 185
DOI: 10.1016/j.bmc.2012.10.044

Target ChEMBL ID: CHEMBL398
ChEMBL Target Name: KB
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_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: CHEMBL739443

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: Bioorg Med Chem Lett
Year: 2000
Volume: 10
Issue: 4
First Page: 323
Last Page: 327
DOI: 10.1016/s0960-894x(99)00702-7

Target ChEMBL ID: CHEMBL375
ChEMBL Target Name: Mus musculus
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: 2152-01_Activator_SinglePoint_HTS_Activity

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

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

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

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

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

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

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

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

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

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

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

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

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

External ID: 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: 2146-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: D54 ERlucT Cell line was obtained from PI in frozen vials.
Thaw vial into T175. Cells are fragile and may take a day or so to acclimate to growth in flask.

Growth Medium:
DMEM, High Glucose, L-Glu 11965-118 Invitrogen
10% FBS , Certified 16000-044 Invitrogen
Pen-Strep (1X)
200 ug/ml G418 10131-027 Invitrogen
Plating Medium:
DMEM, Phenol Red Free, High Glucose 31053-036 Invitrogen (does not contain L-glutamine)
L-Glutamine 1X
10% FBS, Certified 16000-044 Invitrogen
Pen-Strep
NO SELECTION ANTIBIOTIC (G418)

Using automated tissue culture expand T175 flask to triple flask and subsequently scale up to hyper flask (2-3 days between splitting). Estimated 5 hyperflasks needed for one 220 384 well plate run.
Once cells are near confluency seed to 384 well plates.
Seed cells in 384 well white solid bottom plates (Costar 8867BC) at 3,000 cells per well in 30 ul volume in plating medium. (see above: Use phenol red free medium that does not contain selection antibiotic(G418) for seeding cells).
Incubate cells overnight at 37 degrees celsius with 5% CO2.
Pin compounds from compound library to a final concentration of 10 uM in the plate for MLPCN library. Pin positive control (tunicamycin) from sentinel plate to positive control wells for a final concentration of 100 nM.
Incubate cells with compound for a full 24 hours in incubator at 37 degrees celsius with 5% CO2.
Bring plates out of incubator to deck to equilibrate to room tempurature (20 minutes) .
Add 20 ul of Bright-Glo (Promega) reagent (luciferin+ ATP) per well.
Incubate 10 minutes at room temperature.
Read on Envision plate reader using standard luminescence setting , 0.1 sec read 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 'Stimulators Minus 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 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 25.

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

External ID: 2132-01_Agonist_SinglePoint_HTS_Activity

Protocol:
Vibrio cholerae quorum-sensing modulator bioassay
Reporter strain:
1.BH1578 (V. cholerae AcqsA AluxQ carrying pBB1 cosmid, which contains the V. harveyi luxCDABE luciferase operon)
Materials:
LB Medium: Dissolve in 10 g/L Tryptone, 5 g/L Yeast Extract, and 10 g/L NaCl in distilled water, autoclave, store at room temperature
Tetracycline (10 mg/mL): Dissolve 10 mg tetracycline in 1 mL 100% ethanol, store at -20 degrees C, protect from light
LB/tet: add 1 mL tetracycline (10 mg/mL) to 1L of LB medium. Final concentration of tetracycline is 10 microg/mL. Prepare it fresh on a daily basis.
CAI-1 stock: Dissolve CAI-1 in DMSO to 50 mM (10.7 mg/mL), store at -20 degrees C
Procedures:
1.Grow up the BH1578 reporter strain in 100 mL LB/Tet at 30 oC for >16 hours with shaking (200 rpm). The final OD600 of each culture should be > 3.0
2.Dilute the culture to an OD600 of 0.9 with LB/Tet, mix well. (Note: avoid biofilm aggregates in the culture, a low speed centrifugation (200 rpm for 1 min) should remove most aggregates)
3.Add 20 uL of LB-Tet per 384 well assay plate with the Thermo Combi fluid dispenser. Add 150 nL of compound per well.
4.Dispense 10 microL of diluted culture into each well of a 384 well plate (Black with clear bottom Greiner 781096 plates). Compounds are screened at 20 microM final concentration. 1 uM CAI-1 was used as the positive control.
5.Incubate the plates at 30 degrees C for 6 hours.
5.Measure bioluminescence (USLum(384)) and OD600 in a Perkin-Elmer Envison Multilabel Reader

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

EXPECTED OUTCOME: Active compounds result in increasing readout signal.
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (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: 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 30.

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 50.

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 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

NOTE: Raw signal values for inactive compounds were outside the linear detection range of the plate reader; therefore, no REPRODUCIBILITY_COSINE_TRANSFORM was calculated for inactive compounds.

External ID: CHEMBL2318167

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2013
Volume: 21
Issue: 1
First Page: 178
Last Page: 185
DOI: 10.1016/j.bmc.2012.10.044

Target ChEMBL ID: CHEMBL613426
ChEMBL Target Name: Serum
ChEMBL Target Type: TISSUE - Target is a healthy or diseased tissue
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

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: CHEMBL2318165

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2013
Volume: 21
Issue: 1
First Page: 178
Last Page: 185
DOI: 10.1016/j.bmc.2012.10.044

Target ChEMBL ID: CHEMBL375
ChEMBL Target Name: Mus musculus
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

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: CHEMBL2318164

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2013
Volume: 21
Issue: 1
First Page: 178
Last Page: 185
DOI: 10.1016/j.bmc.2012.10.044

Target ChEMBL ID: CHEMBL614073
ChEMBL Target Name: M109
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-A699-DNMT1-Inh-Primary-Assay

Protocol: Assay materials:
1) DNMT1 methyltransferase (catalytic domain 520-1632aa) was obtained from the Dr. Jim Stivers laboratory.
2) 23-mer DNA oligo, methylated and labeled with fluorescein, 5'-FAM-GAG AAG -iMe-dC-GC AGT GGG TGG ATC CAG -3', and 24-mer DNA oligo, conjugated to the dabcyl quencher, 5'- CTG GAT CCA CCC ACT GCG GTT CTC -Dab-3', were custom synthesized by Integrated DNA Technologies
3) Hinp1I restriction endonuclease (Cat # 303-0125) and S-adenosylmethionine (Cat # B9003S) were purchased from New England Biolabs
4) Assay Buffer: 100 mM Tris-HCl pH 8.0 , 2 mM EDTA , 1 mM DTT, 0.004% BRIJ35, 10% Glycerol
5) Stop Solution: 100 mM Potassium Chloride, 11 mM Magnesium Chloride
6) Corning 1536 well black plate (Cat # 3724)

uHTS Procedure

1) Using LabCyte Echo, transfer 37.5 nL of test compounds from a 2 mM compound source plate into assay plate Cols. 5-48 (final concentration of test compounds is 10 uM, 0.5 % DMSO). Transfer 37.5 nL of 100% DMSO into assay plate Col. 1-4.
2) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
3) Using Thermo Scientific MultiDrop Combi, dispense 2 uL of Assay Buffer into columns 1-2, dispense 2 uL of 30 nM DNMT1 solution in Assay Buffer into columns 3-48.
4) Using Thermo Scientific MultiDrop Combi nL, dispense 2 uL of SAM/DNA Mix, containing 3 uM S-adenosylmethionine and 250 nM duplex DNA oligo in Assay Buffer, into columns 1-48.
5) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
6) Incubate for 120 minutes at room temp.
7) Using Thermo Scientific MultiDrop Combi nL, dispense 1.5 uL of Stop Solution into columns 1-48.
8) Using Thermo Scientific MultiDrop Combi nL, dispense 2 uL of 1u/uL Hinp1I into columns 1-48.
9) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
10) Seal the plates and incubate for 16 hours at 37 degrees C
11) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
12) Read plates on Perkin Elmer Envision at Ex/Em 485/520 nM in fluorescence intensity mode

Comment: Compounds that demonstrated an activation of >= 50% at 10uM concentration are defined as actives in this assay.

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

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

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

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

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

External ID: 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: CHEMBL734835

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem. Lett.
Year: 2000
Volume: 10
Issue: 4
First Page: 323
Last Page: 327
DOI: 10.1016/s0960-894x(99)00702-7

Target ChEMBL ID: CHEMBL375
ChEMBL Target Name: Mus musculus
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL4149441

Protocol: N/A

Comment: Journal: J Nat Prod
Year: 2018
Volume: 81
Issue: 5
First Page: 1131
Last Page: 1142
DOI: 10.1021/acs.jnatprod.7b00384

Target ChEMBL ID: CHEMBL614123
ChEMBL Target Name: J774
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: CHEMBL4149442

Protocol: N/A

Comment: Journal: J Nat Prod
Year: 2018
Volume: 81
Issue: 5
First Page: 1131
Last Page: 1142
DOI: 10.1021/acs.jnatprod.7b00384

Target ChEMBL ID: CHEMBL614123
ChEMBL Target Name: J774
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: CHEMBL3050827

Protocol: N/A

Comment: Journal: J Agric Food Chem
Year: 2011
Volume: 59
Issue: 5
First Page: 1673
Last Page: 1682
DOI: 10.1021/jf104742n

Target ChEMBL ID: CHEMBL612399
ChEMBL Target Name: Botrytis cinerea
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

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: SBCCG-A1014-TWEAK-Fn14-Primary-Assay

Protocol: A. Brief Description of the Assay:
This assay attempts to identify small molecule inhibitors of TWEAK-Fn14 interactions, using a cell-based reporter assay. The assay is run in 1536-well format and is measured via luminescence.

B. Assay Procedures:

Day 1#
1.#Harvest cells. Spin, resuspend in 10% FBS DMEM
2.#Split cell at 1:6
3.#Incubate the flasks for 48hr prior to the experiment day in 37 C 5% CO2 incubator.
#
Day 3#
1.#Harvest cells. Spin, resuspend in Opti-MEM and count.
2.#Dilute cell to 0.25 x106 c/mL.
3.#Prime the Multidrop Combi with 5ml Opti-MEM and add 4microL Assay media to full plate.
a.#>>Plate with Multidrop Combi.
4.#Spin plates at 1000 RPM for 1 min, lid plate.
5.#Incubate plate overnight in 37 C 5% CO2 incubator.
#
Day 4#
1.#Transfer compound (10mM) and DMSO using LabCyte ECHO Dose Response protocol.
2.#Spin plates at 1000 RPM for 1 min.
3.#Incubate plate for 1 hour in 37 C 5% CO2 incubator.
4.#Transfer 2microL of Human TWEAK (120ng/ml) using Multidrop Combi.
5.#Spin plates at 1000 RPM for 1 min and incubate plate for 8 hours in 37 C 5% CO2 incubator.
6.#Add 2.5 microL of Bright Glo reagent and spin the plates at 1000rpm for 1min.
7.#Incubate at RT for 5 min.
8.#Read plate with PerkinElmer ViewLux.

C. Plate Map:

Positive (Low) control in column 1-4, DMSO.
Negative (High) control in columns 45-48, DMSO.
Test wells in columns 5-44.

Comment:

External ID: CHEMBL5047503

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J Med Chem
Year: 2022
Volume: 65
Issue: 1.0
First Page: 460
Last Page: 484
DOI: 10.1021/acs.jmedchem.1c01605

Target ChEMBL ID: CHEMBL395
ChEMBL Target Name: HepG2
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: CHEMBL3050831

Protocol: N/A

Comment: Journal: J Agric Food Chem
Year: 2011
Volume: 59
Issue: 5
First Page: 1673
Last Page: 1682
DOI: 10.1021/jf104742n

Target ChEMBL ID: CHEMBL2367277
ChEMBL Target Name: Phomopsis obscurans
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

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: CHEMBL3050835

Protocol: N/A

Comment: Journal: J Agric Food Chem
Year: 2011
Volume: 59
Issue: 5
First Page: 1673
Last Page: 1682
DOI: 10.1021/jf104742n

External ID: CHEMBL5047506

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J Med Chem
Year: 2022
Volume: 65
Issue: 1.0
First Page: 460
Last Page: 484
DOI: 10.1021/acs.jmedchem.1c01605

External ID: CHEMBL4149452

Protocol: N/A

Comment: Journal: J Nat Prod
Year: 2018
Volume: 81
Issue: 5
First Page: 1131
Last Page: 1142
DOI: 10.1021/acs.jnatprod.7b00384

External ID: CHEMBL3039488

Protocol: N/A

Comment: Journal: Mol Pharmacol
Year: 2013
Volume: 83
Issue: 6
First Page: 1257
Last Page: 1267
DOI: 10.1124/mol.112.084152

Target ChEMBL ID: CHEMBL1697668
ChEMBL Target Name: Solute carrier organic anion transporter family member 1B1
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous single protein target assigned

External ID: BHood-p-21Jun2013-01

Protocol: This assay is to identify inhibitors of HNF4 alpha protein binding to DNA
##

A. Materials:##
Description#Company#Cat #
1M TRIS #BioRad#161-0719
DTT#RPI#D11000
Glycerol#Amersco #0854-1L
NaCL#Fisher Bioreagents#BP358-212
Tween 20#Sigma#P1379
HNF4 Protein #Assay Provider#
FITC-DNA#Assay Provider#
1536 well black high base plate#Aurora#00019120BX
##
B. Plate Map:##
Positive control in columns 1 and 2 is DNA and 70nM Protein##
Negative control in columns 3 and 4, Protein at 650nM and DNA, DMSO##
Test compound in columns 5 - 48, Protein at 70nM + DNA + test compound

C. Procedures:##########
Step#Description#########
1#Transfer test compounds to columns 5-48, DMSO to columns 1-4 using ECHO 555.#########
2#Set up Envision as as described in G. Instrument setting.#########
3#Prepare assay buffer.#########
4#Dispense undiluted HNF4 protein solution at into col 3-4 and 140nM HNF4 protein into columns 1,2, and 5-48. #########
5#Dispense 3uL/well of FITC-DNA (FAC = 5nM) using BioRAPTR#########
6#Spin down plates on Eppendorf centrifuge 5810 at 1000 rpm for 1 minute.#########
7#Incubate plates at RT for 1 hour.#########
8#Read using FP protocol on Envision#########
##########
D. Assay Conditions:##########
Reagent##Final Conc.#Unit#######
1#Tris pH7.4#0.02#M#######
2#NaCl#0.05#M#######
3#DTT#0.01#M#######
4#Glycerol#10#%#######
5#Protein#650 for EC100 and 70nM for EC20#uM#######
6#DNA#5#nM#######
*#6uL reaction volume#########
*#60 min reaction at room temp#########
*#10uM test compound#########
*#0.5% DMSO

Comment:

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: 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: CHEMBL4628398

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: Bioorg Med Chem Lett
Year: 2020
Volume: 30
Issue: 8
First Page: 127049
Last Page: 127049
DOI: 10.1016/j.bmcl.2020.127049

External ID: SBCCG-A764-CF-PAF-Primary-Assay

Protocol: Assay Materials:
KKLEB-NFkB-GFP cells (Assay Provider)
PAF(Assay Provider)
Fetal Bovine Serum (Hyclone SH30396.03)
Penicillin Streptomycin solution
L-glutamine (100X)
TrypLE (Invitrogen 12563)
DPBS without calcium and magnesium (1X)
Corning culture flasks
Black CellBind 1536-well plates (Corning 3833)
ATPlite (Perkin Elmer 6016739)

I. Cell Suspension
1- Dispense 3 uL/well of cells at 5X10;5 cells/mL to the whole plate (plate cells in 2% FBS assay media).
2- Spin down plates on Eppendorf centrifuge 5810 at 500 rpm for 1 minute.

II. Compound Addition:
3- Transfer test compounds to columns 5-48 and DMSO to columns 1-4 using the Labcyte ECHO 555.
4- Transfer volume of test compound and DMSO is 15nL, making 5uM compound concentration at 0.25% DMSO final.
5-Spin down plates on Vspin at 1000 rpm for 1 minute.
6-Put Kalypsys metal lids on plates, incubate plates at 37 degrees C with 5% CO2 for 2 hours.

III. Reagent Addition
7- Dispense 3 uL/well of serum free assay media to columns 1 and 2.
8- Dispense 3 uL/well of PAF (dilute in serum free assay media) to columns 3-48.
9- Spin down plates without lids on Vspin at 2000 rpm for 2 min
10- Put Kalypsys metal lids on plates, and incubate plates at 37 degrees C with 5% CO2 overnight.

IV. Reading plates:

11-Spin plates upside down with a container at 1000 rpm for 15 sec. Dab them with a tissue to dry them and Read immediately on envision for GFP fluorescence.
12-Dispense 6 uL/well of ATPlite (diluted in DPBS 1:1).
13-Spin down plates on Eppendorf centrifuge 5810 at 2000 rpm for 2 minutes without lids.
14-Incubate plates for 10 min at RT and run Luminescence read on Viewlux.

Comment: Compounds that demonstrated a corrected %Activity of >= 50% at 5 uM concentration are defined as actives in this assay.

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

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary and single-concentration confirmation screening data.
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30.
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

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

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

External ID: CHEMBL4149440

Protocol: N/A

Comment: Journal: J Nat Prod
Year: 2018
Volume: 81
Issue: 5
First Page: 1131
Last Page: 1142
DOI: 10.1021/acs.jnatprod.7b00384

Target ChEMBL ID: CHEMBL614123
ChEMBL Target Name: J774
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_ANT_FLUO8_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as antagonists and decrease activity of the human M1 muscarinic receptor (CHRM1; M1) that have been pre-treated with a known agonist, with the end result being a decrease in intracellular calcium. In this assay, CHO-K1 cells stably expressing human M1 are loaded with the Fluo-8 calcium indicator dye. Compounds are added followed by treatment with the activator acetylcholine at a concentration that results in 80% activation (Ec80). As designed, compounds that act as CHRM1 antagonists will decrease calcium mobilization, resulting in decreased relative fluorescence of the indicator dye below that of the Ec80 of acetylcholine. 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 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 EC80 concentration of acetylcholine. Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay.

Hits for this assay were determined 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 inhibition was calculated from the median ratio as follows:

%_Inhibition = ( 1 - ( Ratio Test_Compound - Median_Ratio_High_Control ) / ( Median_Ratio_Low_Control - Median_Ratio_High_Control ) ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing Ec80 of acetylcholine and DMSO.
High_Control is defined as wells containing DMSO.

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

The PubChem Activity Score range for active compounds is 100-7, and for inactive compounds 80-0.

In this assay not all plates were run in the same batch. This resulted in batch-to-batch variation among the different batches of plates, thereby necessitating the use of a plate-based activity cutoff. For this reason the inactive and active scores overlap.

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: 250mM (pH 8.0); (Sigma P8761)
Potentiator: 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: FBW7_ACT_ALPHA_1536_1X%ACT PRUN

Protocol: Assay Overview:
FBW7 assay principle. In this assay, either mutant or wild type (w.t.) FBW7 interact with phosphorylated cyclin E peptide (cycE~P), which will bring donor and acceptor beads into close proximity. Laser excitation of the donor beads converts oxygen to an excited singlet state. Reaction of the singlet oxygen with the acceptor beads further activates a chemiluminescence/fluorescence reaction within the same bead resulting in emitted light at 520-620 nm. Small molecule activators that enhance the mutant FBW7 interaction with the cycE~P decrease the distance of the acceptor beads, thus leading to increased signal being emitted signal.
Protocol Summary:
There are six steps in this 1536 well assay format which are listed in order. First, 2.5uL/well of a 2X working solution containing RLFbw7 [12.5nM final], Cyclin E peptide [12.5nM final], and Ni beads [5ug/mL final], in assay buffer (25mM Tris-HCl pH 7.4 + 100mM NaCl, 0.1% Tween-20, 5mM ?-Mercaptoethanol and 0.05% BSA) was dispensed into columns 1-44. Then 2.5uL/well of a 2X working solution containing WTFbw7 [12.5nM final], Cyclin E peptide [12.5nM final], and Ni beads [5ug/mL final], in assay buffer was dispensed into columns 45-48. Using the pintool transfer device 134nL of compound or control was added to each well. This achieved a nominal screening concentration of 26.1uM and 2.6% DMSO concentration. This was followed by the addition of SA beads to all wells at 5ug/mL final concentration in assay buffer. The assay was then incubated for 20 hours in a temperature controlled 25C environment followed by Alphascreen detection using the PerkinElmer EnVision.

The percent activation 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 RLFbw7 (mutant), cyclin E peptide and Nickel acceptor beads in the presence of test compound
High_Control is defined as wells containing WTFbw7 (wild type), cyclin E peptide and Nickel acceptor beads
Low_Control is defined as the median of the wells containing DMSO, RLFbw7 (mutant), cyclin E peptide and Nickel acceptor beads
PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine active compounds. Two values were calculated: (1) the average percent activation of all compounds tested for the screen, 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 percent activation than the cutoff parameter (1.85% in the case here) 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:
Ni Beads- PerkinEmer Lifesciences Cat#6760619R
RLFbw7-Assay Provider
WTFbw7-Assay Provider
Cyclin E peptide-Assay Provider
5M NaCl- Sigma Cat# S6546-1L
Tween20- Fisher Cat# BP337
Tris 1M pH7.4 Research Organics Cat# 9686T
BSA-Sigma Cat#A7030
?-Mercaptoethanol-SigmaM6250
1536-well plates (Corning, part 7254)

Comment: Due to the size of the Scripps Molecular Screening Center 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 Scripps Molecular Screening Center.

External ID: ALD_INH_FLINT_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as inhibitors of fructose bisphosphate aldolase (FBA) of M. tuberculosis, as monitored by loss (oxidation) of NADH in an enzymatic reaction. Fructose bisphosphate aldolase (FBA) catalyzes the conversion of fructose bisphosphate (FB) into the triose product glyceraldehyde 3 phosphate (G3P) in a reversible fashion. The G3P is converted to dihydroxyacetone phosphate (DHAP) by the helper enzyme triose phosphate isomerase (TPI). A second helper enzyme, glycerol phosphate dehydrogenase (GDH), converts the dihydroxyacetone phosphate to glycerol-3-phosphate with the concomitant oxidation of NADH to NAD, and thus the FBA activity is monitored by the reduction of well fluorescence as measured at 450 nm upon excitation at 340 nm. Compounds are tested in singlicate at a final nominal concentration of 1 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 140 ng/mL FBA, 400 nM ZnCl2, 160 uM NADH and the helper enzymes GDH-TPI (4 U/mL) was dispensed into all wells of a 1536-well plate. Next, 10 nL of test compounds were delivered to each well using a PinTool. DMSO only was dispensed to negative control wells whereas a final concentration of 75 nM of reference compound TD3 was dispensed as a positive control. The assay was then initiated by dispensing 5 uL of Buffer A supplemented with 40 uM of FBP substrate. Plates were incubated at room temperature for 20 minutes before fluorescence was measured (Ex. 340 nm; Em. 450 nm) using the ViewLux 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 containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing Compound TD3 (75 nM final)

PubChem Activity Outcome and Score:

Two values were calculated: (1) the average percent inhibition of all test compounds and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter. Any compound that exhibited a greater % inhibition than this cutoff parameter was declared active.

Potential fluorescent compounds that showed a percent inhibition value significantly higher than the one calculated for the High_Control (i.e. greater than the average % inhibition +/- 3 S.D. of the High_Control wells) were removed from the hit-cutoff calculation.

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 100-1, and for inactive compounds 1-0.

List of Reagents:

FBA enzyme (Assay Provider)
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)
TD3 reference control (Assay Provider)
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. 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 fluorescence 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: CHEMBL5047545

Protocol: N/A

Comment: Compounds with activity <= 10uM or explicitly reported as active by ChEMBL are flagged as active in this PubChem assay presentation.

Journal: J Med Chem
Year: 2022
Volume: 65
Issue: 1.0
First Page: 460
Last Page: 484
DOI: 10.1021/acs.jmedchem.1c01605

External ID: SBCCG-A704-MLLE-PAM2-Inh-Primary-Assay

Protocol: Assay Materials:
Hepes (Sigma)
NaCl (Sigma)
EDTA (Gibco 15575)
10% Tween 20 (MP biomedicals ICN10316890)
GST-Mlle protein (Assy Provider)
FITC Pam2 peptide (Assay Provider)
Molecular grade water (CellGro 46-000-CM)
Assay plate: Aurora 1536 Black Solid Bottom (00019120BX)

I. Prepare Solutions:
1- Prepare 1Xassay buffer in molecular grade water (20mM Hepes pH 7.4, 150 mM NaCl, 0.5mM EDTA, 0.05% Tween-20)
2- Prepare 2x protein solution (1.35uM) in assay buffer
3- Prepare 2x substrate solution (0.01uM) in assay buffer
II. Compound Addition:
4- Using LabCyte Echo 555, transfer 60 nL from 2 mM compound source plate into assay plate Columns 5-48 (final concentration of test compounds is 20 uM, 1.0% DMSO), and 60 nL of DMSO to control wells in Columns 1-4.
III. Reagent Addition
5- Dispense 3uL/well of assay buffer in columns 1 and 2 and 3ul/well protein solution in columns 3-48 using BiorapTR.
6- Dispense 3ul/well of FITC-PAM2 substrate into all wells using BiorapTR.
9- Spin down plates without lids on Vspin at 1000 rpm for 1 min
10- incubate plates at room temp for 2 hours.
IV. Reading plates:
11- Read the plate on PerkinElmer-EnVision plate reader FP protocol with dual mirror

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

Compounds that demonstrated % activity of >= 40 and F-ratio between 0.5 and 1.5 at 20 uM in either normalized or corrected results are defined as active in this assay.

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

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

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

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

External ID: SBCCG-A705-H9c2-Inh-Primary-Assay

Protocol: Assay Materials:
H9c2 Embryonic Myocardium Cells (ATCC CRL-1446)
DMEM (Mediatech 10-013-CM)
DMEM (no phenol red) (Mediatech 17-205-CV)
Fetal Bovine Serum (Hyclone KTL33155)
Penicillin Streptomycin solution Gibco (Invitrogen 667539)
L-glutamine (100X ) Gibco (Invitrogen 25030)
Trypsin-EDTA 0.25% Gibco (Invitrogen 25200)
Hydrogen Peroxide (Macron Chemicals V340-04)
T75 Tissue Culture flasks (75cm2) Corning #3276
T225 Tissue Culture flasks (225cm2) Corning #3293
ATP Lite 1-Step (PerkinElmer 6016739)
PBS (Mediatech 21-031-CV)
1536 well white High Base plate (Aurora Biotechnology 00019130)

I. Compound Addition:
1- Using LabCyte Echo 555, transfer 50 nL from 2 mM compound source plate into assay plate Columns 5-48 (final concentration of test compounds is 20 uM, 1.0% DMSO), and 50 nL of DMSO to control wells in Columns 1-4.
II. Cell Suspension
2-Dispense 3 uL/well of cells using Combi at 1.67X10;5 cells/mL (500 cells/well).
3-Spin down plates on Eppendorf centrifuge 5810 at 1200 rpm for 1 minute.
4-Put Kalypsys metal lids on plates, and incubate plates at 37 degrees C with 5% CO2 for 60 min.
III. Reagent Addition
5-Add 2 uL/well of assay medium to columns 1-2 and 2 uL/well of 1mM H2O2 in assay medium to columns 3-48 of the assay plates using Combi.
6-Incubate plates in CO2 incubator for 2 hours.
7-Open the lid and leave the plate at room temp for 10 min.
8-Add 3uL/well of ATP Lite 1 Step mix using Combi.
9-Spin down plates on Eppendorf centrifuge 5810 without the lid at 2000 rpm for 2 minutes.
10-Cover with black lid and leave the plate at 10 minutes at room temp.
IV. Reading plates:
11- Read the plate on PerkinElmer Viewlux.

Comment: Compounds that demonstrated corrected or uncorrected % activity of >= 50 compared to the controls are defined as inhibitors of the reaction.

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

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

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

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

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

External ID: SBCCG-A706-Rpn11-Inh-Primary-Assay

Protocol: Assay materials:
1) Ub4-pepOG protein substrate was provided by Dr. Deshaies laboratory
2) 26S proteasome from human erythrocytes (Cat # PW9310) and Epoxomicin (Cat # BML-PI127-0100) were purchased from BioMol/ENZO Life Sciences
3) Assay Buffer: 50 mM Tris-HCl pH 8.0 , 0.05 mM ATP, 0.05 mM Magnesium Chloride,
1 mM DTT, 0.01% NP-40
4) Epoxomicin Solution: 1.5 uM Epoxomicin in 1x Assay Buffer
5) Aurora 1536 well black high base plate were obtained from Nexus Biosystems (Cat # 00019120BX)

uHTS Procedure

1) Using LabCyte Echo, transfer 40 nL of test compounds from a 2 mM compound source plate into assay plate Cols. 5-48 (final concentration of test compounds is 20 uM, 1 % DMSO). Transfer 40 nL of 100% DMSO into assay plate Col. 1-4.
2) Pre-incubate 20nM 26S proteasome in Epoxomicin Solution at room temperature for 1 hour, then dilute 10-fold in pre-chilled 1x Assay Buffer.
3) Using Beckman BiorapTR dispense 2uL of Assay Buffer in columns 1 and 2, and 2uL of 26S proteasome solution to columns 3-48.
4) Using Beckman BiorapTR dispense 2ul of Ub4-pepOG substrate into all wells (columns 1-48)
5) Spin plates at 1500 rpm for 1 minute on Eppendorf centrifuge 5810.
6) Incubate for 80 minutes at room temp.
7) Read plates on Perkin Elmer Envision with dual mirror at Ex/Em 480/535 nm in fluorescent polarization mode

Comment: Compounds that demonstrated a normalized or corrected inhibition of >= 40% at 20uM concentration are defined as actives in this assay.

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

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

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

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

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

External ID: SBCCG-A754-STEP-Primary-Assay

Protocol: STEP Assay HTS Protocol

A. Brief Description of the Assay:
This assay idenitfies inhibitors of STEP (STriatal-Enriched Phosphatase) enzyme. It is measured via fluorescence in 1536-well plate format.

B. Materials:
Item, Source, Cat #
STEP Enzyme Stock Solution 6.2mg/mL (178uM), Dr. Lutz Tautz, N/A
Bis-Tris, Fisher Sci, BP301-100
Tween 20, Sigma, P1379
DTT, Sigma, D9779
OMFP, Sigma, M2629-100MG
Mol. Grade Water, Mediatech, Inc., 46-000-CM
1536 well black solid flat bottom Non-Binding plate, Corning, 3724

C. Final Assay Conditions:
Reagent, Final Concentration
BIS-TRIS pH 6.0, 50 mM
Tween 20, 0.005 %
DTT, 2.5 mM
STEP, 0.5 nM
OMFP, 25 uM
Final reaction volume, 4 uL/well in 1536 well plate
Test compound concentration, 20 uM
Final DMSO concentration, 1.0%
D. Procedures:
Step#, Description
1. Prepare Reagents as described in sections F. Recipe.
2. Using LabCyte Echo, transfer 40 nL from a plate containing 2 mM test compounds into assay plate Col. 5 - 44 (final concentration of test compounds is 20 uM, 1.0% DMSO). 40nL of DMSO should be transferred to col. 1-4 for control wells.
3. Spin plates at 1000 rpm for 1 minute in centrifuge.
4. Set up Kalypsys dispenser as described in section G. Instrument settings.
5. Using the Kalypsys dispenser, add 2 uL/well of control buffer (no enzyme control) to columns 1 and 2 for the positive control wells.
6. Using the Kalypsys dispenser, add 2 uL/well of enzyme solution to col. 3-48 for the negative control and test compound wells.
7. Using the Kalypsys dispenser, add 2 uL/well of substrate solution to columns. 1-48 (all wells).
8. Spin plates at 1000 rpm for 1 minute in centrifuge.
9. Incubate plates in the dark at room temperature for 20 minutes.
10. Detect signals on Perkin Elmer Viewlux with settings as described in section G. Instrument settings.

E. Plate Map:
Positive (Low) control in columns 1 - 2, DMSO, substrate only
Negative (High) control in columns 3 and 4, DMSO, enzyme and substrate
Test compound in columns 5 - 48, Test compounds, enzyme and substrate

F. Recipe:
Enzyme solution (STEP)
Reagent, Working Conc.
BIS-TRIS pH 6.0, 50 mM
Tween 20, 0.005 %
DTT, 5 mM
STEP, 0.5 nM

Substrate solution (OMFP)
Reagent, Working Conc.
BIS-TRIS pH 6.0, 50 mM
Tween 20, 0.005 %
OMFP, 50uM

G. Instrument settings:
Kalypsys dispenser
Step#, Description
1. Before assay starts, rinse tubing thoroughly with 5 mL of MilliQ H2O per dispensing tip.
2. Air rinse tubing.
3. Rinse and prime tubing with 1 mL of actual reagents per dispensing tip.
4. When the assay is done, clean tubing thoroughly with 5 mL of MilliQ H2O per dispensing tip.
5. Air rinse tubing.
6. Rinse tubing thoroughly with 5 mL of 25% EtOH per dispensing tip.

Perkin Elmer Viewlux
Light Energy: 10000
Measurement: Time 1 sec.
Excitation Filter: 480/20 (FITC)
Emission Filter: 540/25 (FITC)
Mirror: FITC dichroic
Sensitivity: 4.13 e - /ADU

H. Note:
1. All reagents should be made up according to its spec-sheet or otherwise in Mol. Grade Water.
2. Make up buffer minus Tween-20 in large scale and add fresh Tween-20 weekly.
3. Make up enzyme buffer minus DTT in large scale and add fresh DTT just before the assay starts.
4. Storage conditions after reagents are made up:
Reagent, Temp.
Buffer minus DTT, 4 degree
STEP, -80 degrees
Na3VO4, -80 degrees
DTT, -80 degrees
OMFP, -80 degrees (light sensitive)

The experimental values were normalized by difference between values from neutral and stimulator control wells in each plates. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing and edge effect due to overnight incubation. The algorism "Assay Pattern (Multiplicative)" was applied in Genedata Screener(R) software to correct screen data. Further information about data correction is available at http://www.genedata.com/products/screener.html.
Compounds that demonstrated % activity of >= 40 % at 20 uM

Comment: Compounds that demonstrated a normalized or corrected inhibition of >= 40% at 20uM concentration are defined as actives in this assay.

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

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

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.

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

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

External ID: 7071-02_Activator_Dose_CherryPick_Activity_Set2

Protocol: Mammalian Cytotoxicity Assay Panel SOP


Assay Protocol:
Day 0, HepG2 cells were grown in Triple flasks (NUNC) to ~95% confluence (TrypLE Phenol Red free) and resuspended for dispensing at 50,000 cells/mL of DMEM, 10% FBS/Pen/Strep/L-Glutamine (using the TAP Compact SelecT automated cll culture system).

Day 1: Plate cells @2000 per well in 40 microL media (DMEM/10% FBS/Pen/Strep/L-Glutamine) using Corning 8867BC 384 well plates; incubate in standard TC conditions (5% CO2; 95% humidity, 37C) for 24 hours (Compact SelecT).

Day 2: Add 100 nL compound per well at dose into 40 uL assay volume using a pin tool (CyBi Well). Pin 100 nL cytotoxic compounds, mitoxantrone to positive control wells to a final concentration (CID 4212) of 10 uM (100 nL 4 mM DMSO stock). Incubate for 72 hours at 37C in Liconic incubator, 95% humidity 5% CO2.

Day 4: Remove plate from incubator, cool for 15 minutes to room temperature; add 20 uL 50% Promega CellTiter-Glo (diluted 1:1 with PBS, pH 7.4) with Thermo Combi.
Incubate at RT for 5 minutes.

Read plates on a Perkin-Elmer EnVision plate reader with standard luminescence settings for 0.1 sec per well.

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).

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

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

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.

External ID: 2161-01_Activator_Dose_CherryPick_Activity

Protocol: Stock Reagents

Aliquots of 20ug/mL bFGF (Stemgent cat#03-0002-250, 250ug)(HL)-Add 1mL PBS to 250ug bFGF
-Transfer to a conical tube containing 10.5mL PBS
-Add 1mL PBS to tube to wash and transfer contents to conical tube
-Sterile filter, then aliquot in 1mL increments and store in -20 degrees C freezer
OR(BL) Dilute 250 ug bFGF in 12.5 ml PBS for final of 20 ug/ml. Store -20 degrees C freezer.
Aliquots of 20ug/mL EGF (Sigma cat#E9644, 0.2 mg)
-Dissolve 0.2 mg EGF in 1mL DMEM (Gibco 11965-118)-Transfer to 15mL conical, and add 9mL DMEM and mix- Sterile filter, then aliquot in 1mL increments and store in -80 degrees C freezer
OR
(BL) Dilute 0.2 mg in 10ml DMEM. Store in -80 degrees C .
Aliquots of 5ug/mL Heparin (Sigma cat#H3149-purchased by KU-not weight!)
(HL)-Measure 0.22g Heparin in a 50mL conical tube
-Add 10mL Ham's F12 media (MediaTech cat# 10-080-CV) to Heparin powder and mix
-Add additional Ham's F12 to a total of 44mL and mix
-After mixing, let sit at room temperature for a few minutes to completely dissolve the powder
-Sterile filter, then aliquot in 1mL increments and store in 20 degrees C freezer
OR(BL) Dilute 0.22g add 54 ml Ham's F12 media and store in 20 degrees C freezer.
Aliquots of 10mg/mL Poly-ornithine (Sigma cat#P3655-50MG)
(HL)- Add 5mL autoclaved H2O to the bottle containing 50mg powder poly-L-ornithine bromide
-Mix and let sit for a few minutes at room temperature
-Sterile filter, then aliquot in 0.5mL increments and store in -20 degrees C freezer
OR(BL) Add 5 ml sterile H2O to 50mg poly-L-ornithine bromide. Store -20C.

Stock of 0.025 mg/ml Corning Synthemax II-SC Substrate (Corning Cat#3535XX1
(BL) Dilute 10 mg in 400 ml sterile water.
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Media Compositions

Basal NPC media (1L): (BL)
-678mL DMEM (Gibco cat#10313-021, DMEM+High Glucose, Pyruvate, no glutamine)
-291mL Ham's F12 (MediaTech cat#10-080-CV)
-10mL 100X P/S/Q-Final 1%
-20mL B27 (Gibco cat#17504-044)-Final 2%
-Sterile filter

Complete NPC +EFH media:
For 10mL NPC +EFH media, add:
-10uL of 1000XFGF (Final 0.02 ug/ml)
-10uL of 1000X EGF (Final 0.02ug/ml)
-10uL of 1000X Heparin (Final 5 ng/ml)
-0.8uL Puromycin (4uL/50mL) (use for every other passage)
-------------------------------------------------------------------------------------------------------------------------------

Making Wnt3a-conditioned media

- Wnt3a-CM was prepared from mouse L-cells (ATCC #CRL-2648) stably transfected with Wnt3a-expressing vector.

1. Culture L-cells in DMEM basal medium, 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin/glutamine.

2. Allow Wnt3a-secreting L-cells to grow to 90% confluency in multiple culture flasks (Can do in stages, but plan prepare appropriate amount for a full HTS screen). From this point (Day 0):

3. Culture media collected and replaced with fresh media every two days for 3 collections (Days 2, 4 and 6). .

4. When 3 batches of supernatant combined, aliquot and store at -80oC.

5. Spin media at 1000rpm for 5 min to clear debris.

6. Measure collected Wnt3a-CM activity to be used in TCF/LEF reporter assay in dose response curve

7. Calculate EC25 to determine CM concentration to use in assay.
-------------------------------------------------------------------------------------------------------------------------------

Coating Flasks/Plates

Working reagent concentrations:
-Final concentration Poly-ornithine: 20 ug/mL, 1:500 dilution in ddH2O
-Final concentration Laminin: 1:200 in PBS

Coating Flasks:
1. Dispense appropriate amount of Poly-ornithine (1:500) into ddH2O. Dispense coating solution into desired flask size. Keep at room temperature for at least 3 hours.
.T-75 = 20mL
.T-175 = 30mL
.T-225 = 40mL
2. Aspirate poly-ornithine.
3. Dispense appropriate amount of Laminin (1:200) into cold DPBS. Dispense coating solution into flasks.
4. Wrap coated flasks and put into 4C fridge for future use. If using flasks the next day, store them at 37C overnight.

Coating Plates (384-well format):
1. Dispense 20uL/well synthemax/well.
2. Incubate 2hrs r.t. or O.N. at 4C.

-------------------------------------------------------------------------------------------------------------------------------

Passaging/Splitting Cells

1.Aspirate coating from plates

2.Aspirate PBS and add appropriate amount of TrypLE to the flask (see below). Make sure to coat entire surface of cells by rocking back and forth several times.
.T75 - 1.5 mL TrypLE
.T175 - 3mL TrypLE
.T225- 4ml TrypLE

3.Incubate at room temperature for approximately 2 minutes (continually checking under microscope) until cells start to detach from the flask.

4. Add 9 mL basal NPC media (no supplements) to cell solution in flasks to inactivate TrypLE. Wash bottom surface of flask with cell solution (to detach any remaining cells), and then transfer contents of flask to a 50mL conical tube.

5.Centrifuge cells at 1500rpm for 5min.

6.Aspirate supernatant from conical tube and gently re-suspend cell pellet with 10mL basal NPC media using a 10 mL serological pipette. Avoid bubbles if possible.

7.Count Cells - Remove 20uL of 10mL cell suspension and mix with 20uL Trypan Blue in a separate Eppendorf tube. Mix multiple times with pipettor, and transfer 20uL of solution to disposable cell counting slide or do manual counting with hemocytometer.

8.Make appropriate amount of complete NPC media for passaging (see below) by adding supplements (+EFH) to basal NPC media.
.T75 - 20mL complete media/flask
.T175 - 30mL complete media/flask

9.Aspirate the synthemax from the previously coated flasks

10.Transfer desired concentration of cells to new flask containing complete NPC media and label with passage # and other pertinent information.
(Cells were cultivated and expanded in collaborator's lab. Cells were frozen down at 17.5 million cells/vial. For experiments at the Broad, cells are thawed into T225 coated flasks in NPC complete medium and are ready to use for experiments 4-5 days later.)
-------------------------------------------------------------------------------------------------------------------------------

Assay Schedule

Day1:-Harvest cells (refer to cell splitting protocol)
-384w Format; Plate desired number of plates at 6,000 cells/well in 30uL Complete NPC Media on synthemax coated, white walled corning TC plates.
-Incubate for 24 hours at 37C, 5%CO2

Day2:
-Dilute Conditioned media to 20% conditioned media in NPC media.
-Add 10uL conditioned media to each well using Combi (Final conditioned media concentration is 5% in well)*** Variations will need to be empirically determined.
-Incubate for 1 hour at 37C
-Remove plates from incubator
-Pin compound plate+poscon sentinel plates
-Incubate for 24 hours at 37C

Day3:- Remove plates from incubator
-Incubate 10 minutes RT
-Add SteadyGlo to each well using Combi (10uL)
-Incubate for 10 minutes covered at room temperature
-Read on Envision (Protocol: 384-0.1s US Lum)
-------------------------------------------------------------------------------------------------------------------------------

Comment: PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal Max_Concentration.

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

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

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold 30.0
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= 30% but <= 70%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.