External ID: OPRM1-OPRD1_AG_LUMI_1536_1X%ACT PRUN

Protocol: Assay Overview:

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

Protocol Summary:

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

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

The percent activation for each compound was calculated as follows:

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

Where:

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

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

PubChem Activity Outcome and Score:

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

The PubChem Activity Score range for active compounds is 100-2, and for inactive compounds 2-0.

List of Reagents:

PathHuntertrade mark B-arrestin recruitment assay, containing the U2OS OPRM1 OPRD1 Beta-arrestin cell line; PathHunter Detection Kit (DiscoveRx, part, 93-0558C3)
Ham's F-12 media (Invitrogen, part 11765-054)
DMEM media (Invitrogen, part 11995-073)
Detachin (Genlantis, part T100100)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082-147)
Puromycin (Invitrogen, part A11138-02)
Hygromycin B (Invitrogen, part 10687-010)
Geneticin (Invitrogen, part 10131-027)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Nunc, part 159910)
Agonist: Deltorphin B (Anaspec, part 62683)
1536-well plates (Greiner, part 789173)

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

External ID: 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: CHEMBL4311817

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 2
First Page: 442
Last Page: 446
DOI: 10.1016/j.bmc.2018.12.023

Target ChEMBL ID: CHEMBL397
ChEMBL Target Name: Jurkat
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: CHEMBL4311815

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: 2019
Volume: 27
Issue: 2
First Page: 442
Last Page: 446
DOI: 10.1016/j.bmc.2018.12.023

Target ChEMBL ID: CHEMBL614102
ChEMBL Target Name: PC-9
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: CHEMBL4311814

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 2
First Page: 442
Last Page: 446
DOI: 10.1016/j.bmc.2018.12.023

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: CHEMBL4311813

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: 2019
Volume: 27
Issue: 2
First Page: 442
Last Page: 446
DOI: 10.1016/j.bmc.2018.12.023

Target ChEMBL ID: CHEMBL614348
ChEMBL Target Name: NCI-H1975
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: CHEMBL4311824

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 2
First Page: 442
Last Page: 446
DOI: 10.1016/j.bmc.2018.12.023

Target ChEMBL ID: CHEMBL220
ChEMBL Target Name: Acetylcholinesterase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

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: CHEMBL1015630

Protocol: N/A

Comment: Journal: J. Nat. Prod.
Year: 1988
Volume: 51
Issue: 2
First Page: 345
Last Page: 348
DOI: 10.1021/np50056a030

Target ChEMBL ID: CHEMBL3649
ChEMBL Target Name: Xanthine dehydrogenase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4311822

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: 2019
Volume: 27
Issue: 2
First Page: 442
Last Page: 446
DOI: 10.1016/j.bmc.2018.12.023

Target ChEMBL ID: CHEMBL612557
ChEMBL Target Name: RAW264.7
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: 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: CHEMBL4311821

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: 2019
Volume: 27
Issue: 2
First Page: 442
Last Page: 446
DOI: 10.1016/j.bmc.2018.12.023

Target ChEMBL ID: CHEMBL612557
ChEMBL Target Name: RAW264.7
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: 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: 2070-01_Inhibitor_SinglePoint_HTS_Activity

Protocol: Protocol:
ASSAY PROCEDURE FOR HEDGEHOG AUTOPROCESSING

Buffer A: 20 mM sodium phosphate, pH 7.5, 0.5 M NaCl

Buffer B: 20 mM sodium phosphate, pH 7.5, 0.5 M NaCl, 8 M urea
This is prepared by adding 48 g of urea (enzyme grade) to 50 ml of double-strength Buffer A and adjusting volume to 100 ml with H2O.


Buffer D: 20 mM sodium phosphate, pH 7.0, 0.5 M NaCl, and 0.5 M arginine
(2.0 M arginine, pH 7.0, is prepared by dissolving 211 g of L-arginine hydrochloride (obtained from Research Organics) in 300 ml of H2O, adjusting pH to 7.0 with NaOH, and adjusting volume to 500 ml)



REAGENTS:
Urea - American Bioanalytical AB02100
Arginine Hydrochloride - American Bioanalytical AB000164
TCEP [Tris(2-carboxyethyl)phosphine hydrochloride] - Sigma C4706
DM-MEA [2-(Dimethylamino)ethanethiol] - Aldrich D141003 (very hygroscopic)
Cholesterol - Sigma C8667
FLAsH-EDT2-Invitrogen T34561

CELL DISRUPTION:

Suspend the cells from 50 ml of E. coli [Rosetta 2 (DE3)/pET45Dmel710#2] in 3 ml of Buffer A +
1 mM PMSF and disrupt by passing twice through the small French pressure cell. Centrifuge in 12 ml Sorvall tubes at 12,000 rpm for 30 min. and collect the pellet (inclusion bodies). The pellet is washed twice by resuspending in 4 ml of Buffer A + 1 mM PMSF and centrifugation at 12,000 rpm for 20 min. The pellet is then then resuspended in 3 ml of Buffer B + 1 mM PMSF to extract the inclusion bodies and centrifuged at 12,000 rpm for 20 min. Save the supernatant and extract the pellet a second time with 3 ml of Buffer B + 1 mM PMSF and combine the supernatants. Centrifuge the combined supernatants in the Sorvall at 18,000 rpm for 60 min to remove insoluble material and yield the solubilized inclusion bodies (IB). Save inclusion bodies at 4 degrees C after diluting about 10% with Buffer A to avoid crystallization of urea.

Typically, the protein concentration of the inclusion bodies is 1 mg/ml. The material is stable on storage at 4 degrees C for at least a month and can be kept frozen at -80 degrees C in 1 ml portions indefinitely.


Dilution Buffer:
297 ml of Buffer D
3 ml of 0.1 M TCEP
300 ml Final

Substrate mix
6.5 ml of 26 mM cholesterol in 95% EtOH
2.0 ml of 1% Triton X-100
41.5 ml of H2O
50 ml + 200 ml Dilution Buffer (cholesterol = 680 microM) - Dilute immediately before use

Control mix
0.8 ml of 1% Triton X-100
19.2 ml of H2O
20 ml + 80 ml Dilution Buffer (No cholesterol)

IB mix
13.2 ml Hedgehog protein ( approximately 1.65 mg/ml) [12 ml form prep of 10.05.11, 2 ml from prep of 10.05.10]
198 ml Buffer D (
room temp)
2.2 ml of 0.1 M TCEP
2.2 ml of 0.1 M DM-MEA
2.2 ml of 0.17 mM FlAsH
2.2 ml of H2O
220 ml final




Incubate at 25*C for 2 h. Dialyze against 3 X 1 liter Buffer D + 1 mM TCEP (10 ml of 0.1 M TCEP per liter) for 6 h - 14h. Recommended dialysis tubing: SpectraPor MWCO 8,000, flat width = 12 mM (VWR 25223-650). Readjust volume to 220 ml after dialysis.The dialyzed Hh Protein Cocktail can be stored at 4 degrees C for at least 5 days with negligible loss of activity. It should be stable at room temperature for at least 12 h.




Dispense 35 microl per well. Add IB mix first to entire plate. Wait 30 min before adding cholesterol, while control mix (35ul) is added to columns 23&24.

Then add substrate mix to columns 1-22, 40 min after adding protein. Fluorescence polarization was measured after 1 hour incubation at 25C using Tecan Safire 2 microplate reader (Tecan, Mannedorf, Switzerland) with excitation at 390 nm and emission at 550
nm.

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

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

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

PATTERN CORRECTION: 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 to be three standard deviations above the mean neutral control value.

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 2 (active) :
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

External ID: CHEMBL1002694

Protocol: N/A

Comment: Journal: J. Nat. Prod.
Year: 1979
Volume: 42
Issue: 1
First Page: 85
Last Page: 91
DOI: 10.1021/np50001a002

Target ChEMBL ID: CHEMBL389
ChEMBL Target Name: P388
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: CHEMBL1002689

Protocol: N/A

Comment: Journal: J Nat Prod
Year: 1979
Volume: 42
Issue: 1
First Page: 85
Last Page: 91
DOI: 10.1021/np50001a002

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: SMAD3201

Protocol: Suspensions of trypsinized HEPG2 CAGA-GFP cells were dispensed into white, tissue culture-treated, solid 1536-well plates at 5uL/well (1000 cells/well final concentration) in DMEM medium supplemented with 1% FBS. Plates were incubated at 37 degrees C for 2 hours, after which 23 nL of compounds or DMSO were delivered to each well using a pin tool. One uL of recombinant TGF-beta in DMEM (1% FBS) was then dispensed (500 pg/mL final concentration), and plates were incubated at 37 degrees C for 18 hours. Two uL of CellTiter Glo (Promega), a luminescence-based viability reagent, was dispensed, followed by a 10 minute room temperature incubation. The plates were then measured on a PerkinElmer ViewLux plate reader for luminescence (clear filter) using a 5 second exposure. The %Activity was determined from the corrected luminescence values. Wells containing media only (no cells) were used to normalize %Activity of identified toxic compounds; media-only wells corresponded to 100%Activity (complete cell-killing), while DMSO-dosed cell controls were used to normalize 0%Activity (no toxicity).

Concentration-response curves were fitted to the signals arising from the resulting luminescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Toxic compounds showed concentration-dependent decreases in luminescence, concordant with a decrease in intracellular ATP concentration (CellTiter Glo's marker of viability), and thus a decrease in the number of viable cells. Inactive (non-toxic) compounds showed no effect on luminescence signal. Active (toxic) compounds showed concentration dependent decrease in luminescence.

Comment: Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description".

2. For all inactive (non-toxic) compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active (toxic) compounds, a score range was given for each curve class type given above. Active (toxic) compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

External ID: 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: 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: FLUC100

Protocol: NCGC Assay Protocol Summary:

Reagents: 50mM Tris acetate, pH 7.5; 10mM Mg acetate; 10uM D-luciferin (Sigma #L9504); 10uM ATP; 0.01% Tween-20; 0.05% BSA; 10nM P. pyralis luciferase (Sigma #L9506)
Control compounds used were two known firefly luciferase inhibitors (compounds (2) and (5) in Auld et al., 2010), and DMSO.

Assay Summary:
Three microliters containing firefly luciferase substrates in buffer (final concentrations: 50mM Tris acetate, pH 7.5, 10mM Mg acetate, 0.01% Tween-20, 0.05% BSA, 10uM D-luciferin, and 10uM ATP) are dispensed into each well of a Greiner white, solid-bottom 1536-well format plate using a flying reagent dispenser (FRD). These assay plates were then treated with 23nL of compound or DMSO using a Kalypsys pin tool, which allows for delivery of a 6-point interplate titration of each compound to the assay plate (quantitative HTS), with a final compound concentrations ranging from approximately 60muM to 7pM. One microliter of firefly luciferase in 500mM Tris-acetate buffer was then delivered by FRD to each well for a final enzyme concentration of 10nM. Luciferase activity was then measured using a ViewLux CCD imager (PerkinElmer), with an average exposure time of 2-30 seconds (2X binning, medium/high gain).

Keywords: NIH Roadmap, MLPCN, MLSMR, qHTS, miR-21, firefly luciferase, FLuc, miRNA.

Comment: Compound Ranking:

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

External ID: SMAD3101

Protocol: Suspensions of trypsinized HEPG2 CAGA-GFP cells were dispensed into white, tissue culture-treated, solid 1536-well plates at 5uL/well (1000 cells/well final concentration) in DMEM medium supplemented with 1% FBS. Plates were incubated at 37 degrees C for 2 hours, after which 23 nL of compounds or DMSO were delivered to each well using a pin tool. One uL of recombinant TGF-beta in DMEM (1% FBS) was then dispensed (500 pg/mL final concentration), and plates were incubated at 37 degrees C for 18 hours. The plates were measured on an Acumen eX3 Explorer plate reader for GFP fluorescence (ex488/em500-530). GFP values were calculated by determining the mean GFP fluorescence of individual cells, and compiling these values for each well to determine a total well GFP signal. The %Activity was determined from the corrected fluorescence values. A titration of the known TGF-B inhibitor SB431542 was included to monitor plate performance, while unstimulated HEPG2 (-TGF-B) control wells were used to normalize %Activity of identified inhibitors; unstimulated wells corresponded to 100%Activity (full inhibition), while stimulated cell controls (+DMSO) were used to normalize 0%Activity (no inhibition).

Concentration-response curves were fitted to the signals arising from the resulting fluorescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Active inhibitors showed concentration-dependent decreases in GFP fluorescence, concordant with a decrease in TGF-B/SMAD3-driven GFP expression. Inactive compounds showed no effect on fluorescence signal.

Comment: Compound Ranking:

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

External ID: CHEMBL3807384

Protocol: N/A

Comment: Journal: J. Nat. Prod.
Year: 2016
Volume: 79
Issue: 2
First Page: 293
Last Page: 299
DOI: 10.1021/acs.jnatprod.5b00851

Target ChEMBL ID: CHEMBL1936
ChEMBL Target Name: Stem cell growth factor receptor
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: TTR_ACT_LUMI_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify new small molecules which specifically enhance the synthesis of neuronal Transthyretin (TTR) transcription without increasing hepatic or peripheral TTR levels. In this assay, SHSY5Y human neuroblastoma cells stably expressing the Gaussia luciferase (eGLuc2) gene under the control of 2kb of the human TTR promoter (SHSY5Y TTR eGLuc2) are incubated with test compounds for a defined period, followed by the addition of luciferase substrate followed by a luminescence measurement. As designed, test compounds that activate neuronal TTR levels, will increase well luminescence. Compounds are tested in singlicate at a final nominal concentration of 16.7 uM.

Protocol Summary:

The stably transfected SHSY5Y-TTR neuroblastoma cells were routinely cultured in T-225 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of DMEM supplemented with 10% v/v Fetal Bovine Serum, 500ug/mL Geneticin and 1X Penicillin-Streptomycin-Glutamine.

Prior to assay, cells were suspended to a concentration of 500,000 cells/mL in DMEM containing 10% v/v Fetal Bovine Serum and 1X Penicillin-Streptomycin-Glutamine. The day before the assay, 3uL of Recombinant Lucia Luciferase Protein (20ng/mL final concentration) was dispensed into column 3 only and 1500 cells in 3 uL of cell assay media were seeded into the remaining wells. Next, 51 nL of test compound in DMSO, F11 or FD (167 uM final concentration) in DMSO, or DMSO alone (1.67% final concentration) were dispensed to the appropriate wells. The plates were then incubated for 24 hours at 37 C, 5% CO2, and 95 % RH.

Following the one day incubation, plates were equilibrated to room temperature for 10 minutes. The assay was started by adding 3 uL of QUANTI-Luc detection reagent (prepared according to the manufacturer's protocol) to all wells. Plates were centrifuged and well luminescence was read on a ViewLux microplate reader (Perkin Elmer, Turku, Finland).

The percent activation for each compound was calculated as follows:

%_Activation = 100 * ( ( RLU_Test_Compound - Median_RLU_Low_Control ) / ( Median_RLU_High_Control - Median_RLU_Low_Control ) ) )

Where:

Test_Compound is defined as wells containing cells, test compounds and DMSO.
High_Control is defined as wells containing LUCIA 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 activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.

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 100-7, for inactive 7-0.

List of Reagents:

TTR-SHSY5Y cells (supplied by Assay Provider)
DMEM (Cellgro, part 10-013-CV)
Fetal Bovine Serum (Cellgro, part 35-010-CV)
Penicillin-Streptomycin-Glutamine (100X) (Life Technologies, part 10378-016)
Recombinant Lucia Luciferase Protein (Invivogen, part rec-lucia)
QUANTI-Luc Detection Reagent (Invivogen, part rep-qlc)
F11 (Activator Reference Control, Fisher-Maybridge, part SPB05942SC)
FD (Inhibitor Reference Control, Sigma, part F8257)
Trypsin-EDTA (Life Technologies, part 25200-114 )
Geneticin (Life Technologies, part 10131-027)
T-225 Flasks (BD, part 353138)
1536-well plates (Greiner, part 789173)

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

External ID: CHEMBL3807385

Protocol: N/A

Comment: Journal: J. Nat. Prod.
Year: 2016
Volume: 79
Issue: 2
First Page: 293
Last Page: 299
DOI: 10.1021/acs.jnatprod.5b00851

Target ChEMBL ID: CHEMBL1936
ChEMBL Target Name: Stem cell growth factor receptor
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: SRC3_INH_LUMI_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as inhibitors of the steroid receptor coactivator 1 (SRC3), also known as nuclear receptor coactivator 3 (NCOA3). In this assay, HEK293 cells are transfected with a GAL4-responsive reporter plasmid (pGL4.31, Promega) and an expression vector encoding SRC-3 fused to the DNA-binding domain of GAL4(pBIND-SRC-3). The ability of compounds to reduce coactivator transcriptional activity is assessed by measuring luciferase expression from the reporter gene plasmid. As designed, compounds that inhibit SRC3 ability to induce transcription will lead to a decrease in expression of the luciferase gene, resulting in reduced well luminescence. Compounds are tested in singlicate at a final nominal concentration of 3.6 uM.

Protocol Summary:

Seven million HEK293 cells were seeded into T-175 flasks containing 23 mLs of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v Anti-Anti. Flasks were then incubated for 48 hours at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to the assay, cells were harvested using TrypLE, resuspended in fresh media at a density of 1 million cells per mL and seeded into new T-175 flasks (23 mL per flask). After allowed to attach for one hour at 37 C, 5% CO2 and 95% RH, cells were transfected with 1 mL of preincubated mix of serum-free OptiMEM containing 23 ug of the pGL4.31 reporter plasmid, 2.3 ug of pBIND-SRC3 vector, and 80 uL of transfection reagents. Twenty four hours post transfection, cells were harvested using 5 mL of TrypLE and resuspended at a concentration of 750,000 cells per mL in phenol-red free DMEM media supplemented as described above.

The assay was started by dispensing 5 uL of cell suspension into each well of a white, solid-bottom 1536-well plate using a flying reagent dispenser (3,750 cells per well). The first two columns received cells transfected with reporter plasmid and an empty pBIND vector as a control for background luminescence. Cells were then treated with 18 nL/well of test compounds, DMSO as a negative control (final concentration 0.36%), or Gossypol as a positive control (36 uM final) using a PinTool transfer unit (GNF). Plates were then placed in the incubator at 37 C, 5% CO2 and 95% RH. Twenty four hours later, plates were removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by adding 5 uL per well of ONE-Glo luciferase detection reagent. After a 15 minute incubation time, light emission was measured using the ViewLux plate reader (PerkinElmer).

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

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

Where:

Test_Compound is defined as wells containing test compound treated cells.
Positive_Control is defined as wells containing Gossypol treated cells.
Negative_Control is defined as wells containing DMSO treated cells.

PubChem Activity Outcome and Score:

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

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

The PubChem Activity Score range for active compounds is 100-64, and for inactive compounds 64-0.

List of Reagents:

HEK-293 cells (ATCC, part CRL-1573)
DMEM media (Invitrogen, part 11965)
Fetal Bovine Serum (Hyclone, part SH30088.03)
Anti-Anti (Gibco, part 15240)
TrypLE (Invitrogen, part 12604)
T-175 flasks (Falcon, part 353112)
pGL4.31 (Promega, part C935A)
pBIND-SRC3 (Assay Provider)
TransIT 293 transfection reagent (Mirus Corporation, part MIR-2700)
ONE-Glo luciferase reagent (Promega, part E6130)
White, solid-bottom 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. 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, cytotoxic compounds, compounds that perturb the UAS/GAL4 reporter system, and compounds that quench, inhibit, stabilize, or emit luminescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: SRC1_INH_LUMI_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as inhibitors of the steroid receptor coactivator 1 (SRC1), also known as nuclear receptor coactivator 3 (NCOA1). In this assay, HEK293 cells are transfected with a GAL4-responsive reporter plasmid (pGL4.31, Promega) and an expression vector encoding SRC1 fused to the DNA-binding domain of GAL4 (pBIND-SRC-1). The ability of compounds to reduce coactivator transcriptional activity is assessed by measuring luciferase expression from the reporter gene plasmid. As designed, compounds that inhibit SRC1 ability to induce transcription will lead to a decrease in expression of the luciferase gene, resulting in reduced well luminescence. Compounds are tested in singlicate at a final nominal concentration of 3.6 uM.

Protocol Summary:

Seven million HEK293 cells were seeded in T-175 flasks 23 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v Anti-Anti. Flasks were then incubated for 48 hours at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, cells were harvested using TrypLE, resuspended in fresh media at a density of 1 million cells per mL and seeded into new T-175 flasks (23 mL per flask). After being allowed to attach for one hour at 37 C, 5% CO2 and 95% RH, cells were transfected with 1 mL of preincubated mix of serum-free OptiMEM containing 23 ug of pGL4.31 reporter plasmid, 2.3 ug of pBIND-SRC1 vector and 80 uL of transfection reagents. Twenty four hours post transfection, cells were harvested using 5 mL of TrypLE and resuspended at a concentration of 750,000 cells per mL in phenol-red free DMEM media supplemented as described above.

The assay was started by dispensing 5 uL of cell suspension into each well of a white, solid-bottom 1536-well plate using a flying reagent dispenser (i.e. 3,750 cells per well). The first two columns received cells transfected with the reporter plasmid and an empty pBIND vector as a control for background luminescence. Cells were then treated with 18 nL/well of test compounds, DMSO as a negative control (final concentration 0.36%) or Gossypol as a positive control (36 uM final) using a PinTool transfer unit (GNF). Plates were then placed in the incubator at 37 C, 5% CO2 and 95% RH. Twenty four hours later, plates were removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase was detected by adding 5 uL per well of ONE-Glo luciferase detection reagent. After a 15 minutes incubation time, light emission was measured with the ViewLux reader (PerkinElmer).

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

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

Where:

Test_Compound is defined as wells containing test compound treated cells.
Positive_Control is defined as wells containing Gossypol treated cells.
Negative_Control is defined as wells containing DMSO treated cells.

PubChem Activity Outcome and Score:

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

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

The PubChem Activity Score range for active compounds is 100-63, and for inactive compounds 63-0.

List of Reagents:

HEK-293 cells (ATCC, part CRL-1573)
DMEM media (Invitrogen, part 11965)
Fetal Bovine Serum (Hyclone, part SH30088.03)
Anti-Anti (Gibco, part 15240)
TrypLE (Invitrogen, part 12604)
T-175 flasks (Falcon, part 353112)
pGL4.31 (Promega, part C935A)
pBIND-SRC1 (Assay Provider)
TransIT 293 transfection reagent (Mirus Corporation, part MIR-2700)
ONE-Glo luciferase reagent (Promega, part E6130)
White, solid-bottom 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. 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, cytotoxic compounds, compounds that perturb the UAS/GAL4 reporter system, and compounds that quench, inhibit, stabilize, or emit luminescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

External ID: UNMCMD_DSG_PRIMARY_MLPCN

Protocol: Protocol:
1) Protein G beads (1.06 million beads per 384 well plate) are coupled with the DSG3 antigen by overnight incubation with a cell lysate containing an Fc-DSG3 construct.
2) Coupled beads are used at 3000 beads per well
3) scFv-GFP reagent is diluted in Assay buffer (PBS with 1mM CaCl2, 0.05% BSA, 0.01% Na Azide), added to 382 well assay plates, and incubated with a 20 microM solution of test compounds for 60 minutes at RT.
4) Vehicle control (2%DMSO) and Blocking control (1/40 dilution of soluble DSG3 antigen) are similarly incubated with scFv-anti-DSG3-GFP
5) Pre-coupled beads are added to each well and plates are incubated for 60 minutes with rotation
6) scFv-anti-DSG3-GFP binding to beads is detected using flow cytometry and reported as the Median Channel Fluorescence

Calculations:
Z and Z' values were calculated individually for all plates, most plates passed a Z'>0.3.
An average response value was computed for each plate. Compounds were considered active if the associated well fluorescence was greater than 3SD below the Average Median Fluorescence of the individual plate.

dif = PLATE_CUTOFF - RESPONSE

If diff < 0
Then PUBCHEM_ACTIVITY_SCORE = 0
Else If diff > 100
Then PUBCHEM_ACTIVITY_SCORE = 100
Else
PUBCHEM_ACTIVITY_SCORE = diff

If (PUBCHEM_ACTIVITY_SCORE > 0) AND (RESPONSE > 0)
THEN PUBCHEM_ACTIVITY_OUTCOME = 2 (or ACTIVE)
If (PUBCHEM_ACTIVITY_SCORE > 0) AND (RESPONSE = 0)
THEN PUBCHEM_ACTIVITY_OUTCOME = 3 (or INCONCLUSIVE)
Else
PUBCHEM_ACTIVITY_OUTCOME = 1 (or INACTIVE)

Comment: This reference is not indexed in PubChem
1. Stanley, J.R. 2008. Pemphigus. In Fitzpatrick's Dermatology in General Medicine. K.Wolff, Goldsmith,L.A., Katz,S.I., Gilchrest,B.A., Paller,A.S., and Leffell,D.J., editors. McGraw-Hill. New York. 459-468.

External ID: CHEMBL999161

Protocol: N/A

Comment: Journal: J. Nat. Prod.
Year: 1979
Volume: 42
Issue: 1
First Page: 85
Last Page: 91
DOI: 10.1021/np50001a002

External ID: CHEMBL999162

Protocol: N/A

Comment: Journal: J. Nat. Prod.
Year: 1979
Volume: 42
Issue: 1
First Page: 85
Last Page: 91
DOI: 10.1021/np50001a002

Target ChEMBL ID: CHEMBL386
ChEMBL Target Name: L1210
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: CHEMBL999160

Protocol: N/A

Comment: Journal: J. Nat. Prod.
Year: 1979
Volume: 42
Issue: 1
First Page: 85
Last Page: 91
DOI: 10.1021/np50001a002

Target ChEMBL ID: CHEMBL614027
ChEMBL Target Name: CCRF S-180
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: CHEMBL4136590

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 Nat Prod
Year: 2018
Volume: 81
Issue: 1
First Page: 124
Last Page: 130
DOI: 10.1021/acs.jnatprod.7b00747

Target ChEMBL ID: CHEMBL614793
ChEMBL Target Name: L6
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: CHEMBL4136591

Protocol: N/A

Comment: Journal: J Nat Prod
Year: 2018
Volume: 81
Issue: 1
First Page: 124
Last Page: 130
DOI: 10.1021/acs.jnatprod.7b00747

External ID: CHEMBL4136589

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 Nat Prod
Year: 2018
Volume: 81
Issue: 1
First Page: 124
Last Page: 130
DOI: 10.1021/acs.jnatprod.7b00747

Target ChEMBL ID: CHEMBL612348
ChEMBL Target Name: Trypanosoma brucei rhodesiense
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: PLK1100

Protocol: Four microliter of 25 nM FITC-p-9-mer + 60 nM Plk1 PBD in buffer solution (final concentrations 25 nM and 60 nM respectively) or 4 uL of 25 nM FITC-p-9-mer (final concentration 25 nM) in buffer will be dispensed into a 1,536 Greiner medium-binding black solid plate. Small molecules at final concentrations of 18 nM to 57 uM and positive control phospho-9-mer (sequence: Ac-PPLHSpTAI-NH2) at final concentrations 26 nM to 57 uM and phospho-13-mer (sequence: Ac-CETFDPPLHSpTAI-NH2) at final concentrations of 1 nM to 2.3 uM will be pin-transferred (23 nL) to the plate via a Kalypsys pin-tool robotics equipped with a 1,536-pin array. The reaction plate will be centrifuged for 1 min at 1000 rpm and incubated at room temperature for 10 min. The FITC fluorescence signals will be read using the PerkinElmer ViewLux plate reader at 480 / 20 nm excitation and 540 / 20 nm S and P polarization emission wavelengths. Reagent bottles will be kept submerged into 4 oC recirculating chiller bath and all liquid lines will be covered with aluminum foil to minimize degradation. Library plates will be screened starting from the lowest to the highest concentrations to minimize compound carryover. Vehicle only plates, with DMSO being pin-transferred instead of compound solutions, will be included regularly throughout the screen to record any systematic shifts in assay signal.

Comment: Compound Ranking:

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

External ID: SBCCG-A686-Gli-Sufu-Antagonist-Primary-Assay

Protocol: Gli Antagonist Primary Screen Procedure

Cell Culture:

Media:

Sufu-KO-LIGHT cell line GROWTH MEDIUM
Final concentrations
DMEM Phenol Red containing (Hyclone #SH30243.02)
HI-FBS characterized (Hyclone SH30396.03 HI or equivalent) 10%
L-glutamine (Cellgro # 25-005-CI) 200 mM 100 mL 2 mM (1:100)
Penn/Strep (Cellgro #30-0020CI) 5000 IU/mL 100 mL 50 IU/mL (1:100)
Zeocin (Invitrogen R25005 or Sigma 46-0072) 5 g in 50 mL (100 mg/mL) 0.15 mg/mL (1:666.67)

Sufu-KO-LIGHT cell line ASSAY MEDIUM
Final concentrations
DMEM phenol red free (Hyclone SH30585.02)
HI-FBS characterized (HYCLONE SH30396.03 HI or equivalent) 10%
L-glutamine (Cellgro # 25-005-CI) 200 mM 100 mL 2 mM (1:100)
Na-pyruvate (Sigma S8636-100ML) 100 mM 1 mM (1:100)
Penn/Strep (Cellgro #30-0020CI) 5000 IU/mL 100mL 50 IU/mL(1:100)
HEPES (Omega Scientific # HB-20) 100 mL 1 M 25 mM (1:40)
Zeocin (Invitrogen R25005 or Sigma 46-0072) 5g in 50 mL (100 mg/mL) 0.15 mg/mL (1:666.67)

Other Reagents:

Sufu-KO-LIGHT cell line (Assay Provider)
PBS (Phosphate Buffered Saline)
TrypLE (c) Express cell dissociation reagent (Life Technologies)
T225 tissue culture flasks (Corning)
HYPERflasks (c) (Corning)
1536 well tissue culture plates Aurora (c) & Corning
1536 well Echo (c) compatible Cyclic Olefin Copolymer (COC) compound storage plates (Corning or Labcyte)
384 well low volume Echo (c) compatible COC compound storage plates (labcyte)
Bright-Glo (c) luciferase detection reagent (Promega)

Automation & Instrumentation:

HighRes Biosolutions (HRB) MicroStar robotics platform with Cellario (c) scheduling software integrating the following instruments:
Viewlux (c) microplate imager (PerkinElmer)
VSpin (c) microplate centrifuge (Velocity11/Agilent)
Multidrop Combi liquid handler/dispenser (Thermo)
Liconic tissue culture incubator (Liconic)
Echo (c) 550 acoustic liquid handler (Labcyte)

Other instrumentation:
Biotek Microflo Select (c) peristaltic liquid handler/dispenser
Thermo Centra CL2 Clinical Centrifuge
Nexcelom Bioscience Cellometer (c) Auto T4 cell counter
Eppendorf 5810 centrifuge

Assay Procedure:

Day 1
1) Cells harvested from 2 hyperflasks at 80-90% confluency per screening day.
2) Cells suspended in Sufu-KO-LIGHT assay medium to a density of 1.0e6 cells/mL
3) 5 uL/well cell suspension was dispensed to columns 3-48. 5 uL/well assay medium alone (without cells) was dispensed to columns 1-2 (positive control) in Corning white polystyrene tissue treated 1536 well assay plates (#3727) or Aurora (c) white polystyrene tissue treated 1536 well Low Base square well assay plates (#00029846) using Biotek Microflo Select (c) peristaltic liquid handler/dispenser.
4) Plates were centrifuged 1 min at 1000 rpm (200xG) on an Eppendorf 5810 centrifuge
5) Plates were covered with Kalypsys brand stainless steel assay plate lids and placed over night (16-18 hrs) in humidified Liconic brand automated tissue culture incubator. Plates were stacked vertically in towers which rotate intermittently within the incubator. 37 oC, 5% CO2

Day 2
1) Kalypsys stainless steel lids were removed and 2.5 nL MLSMR test agents at 10 mM in DMSO stored in Labcyte Echo (c) compatible Corning 1536 well Cyclic Olefin Copolymer (COC) plates were applied to assay wells (columns 5-48) using a Labcyte Echo (c) 550 acoustic liquid handling system. Final assay concentration of test agents in the assay was 5 uM. 2.5 nL DMSO controls were also added to control wells (columns 1-4). Final DMSO concentration in the assay was 0.05%.
2) Plates were centrifuged 1 min at 1000 rpm (200xG) on an Eppendorf 5810 centrifuge
3) Plates were covered with Kalypsys brand stainless steel assay plate lids and place over night (16-18 hrs) in humidity controlled Liconic Automated tissue culture incubator. Incubator stacks plates vertically in towers. 37 oC, 5% CO2

Day 3
1) Kalypsys stainless steel lids were removed and replaced with plastic assay plate lids from Corning 1536 well assay plates (#3727) and plates returned to incubator.
2) 3 uL /well Bright-Glo (c) luciferase detection reagent (Promega) was added to assay wells using Multidrop Combi liquid handler (Thermo) and immediately centrifuged for 30 seconds on a VSpin (c) integrated microplate centrifuge at 1500 rpm (Velocity11/Agilent Technologies) and incubated for 10 minutes uncovered at room temperature. NB: plastic assay lids were removed by the HRB robotic system before addition of Bright-Glo (c) and NOT replaced. Luminescence was read on a Viewlux (c) microplate imager (PerkinElmer). Read time: 300 sec, Binning: 4X.

Comment: Compounds that demonstrated %activity >= 55% are defined as actives 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: 7017-01_Other_SinglePoint_HTS_Activity

Protocol: Protocol:

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

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

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

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

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

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

DPBS NaCl
137 mM NaCl

DPBS NaI
145 mM NaI

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

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

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


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

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 8.

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

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

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

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

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

External ID: FIBRIN-TN7_INH_FP_1536_1X%INH PRUN

Protocol: Assay Overview:

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

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


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

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing Aha-Tn7 peptide.
Low_Control is defined as wells containing DMSO.
Sample_Field is defined as wells containing Test Compounds


PubChem Activity Outcome and Score:


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

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

The activity score range for active compounds is 100-15, for inactive 15-0.

List of Reagents:
Fibrin-DD(E ) (Supplied by Assay Provider)
FITC-Tn7 peptide (Supplied by Assay Provider)
Aha-Tn7 peptide (Supplied by Assay Provider)
TRIZMA(R) Base BioXtra >99% (Sigma-Aldrich, part T6791)
Tritontrade mark X-100 BioXtra (Sigma-Aldrich, part T9284)
Sodium Chloride BioXtra >99.5% (Sigma-Adrich, part S7653)
Calcium Chloride Dihydrate BioXtra >99% (Sigma-Aldrich, part C5080)
1536 well plate (Corning, part # 7261)

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

External ID: FIBRIN-TN6_INH_FP_1536_1X%INH PRUN

Protocol: Assay Overview:

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

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



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

Where:

Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing EP-2104R peptide.
Low_Control is defined as wells containing DMSO.
Sample_Field is defined as wells containing Test Compounds

PubChem Activity Outcome and Score:

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

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

The activity score range for active compounds is 100-1, for inactive 1-0.

List of Reagents:

Fibrin-DD(E ) (Supplied by Assay Provider)
TRITC-Tn6 peptide (Supplied by Assay Provider)
EP-2104R peptide (Supplied by Assay Provider)
TRIZMA(R) Base BioXtra >99% (Sigma-Aldrich, part T6791)
Tritontrade mark X-100 BioXtra (Sigma-Aldrich, part T9284)
Sodium Chloride BioXtra >99.5% (Sigma-Adrich, part S7653)
Calcium Chloride Dihydrate BioXtra >99% (Sigma-Aldrich, part C5080)
1536 well plate (Corning, part # 7261)

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

External ID: LXR_ACT_LUMI_1536_3X%ACT for hcDAF-12 AG

Protocol: Assay Overview:

The purpose of this assay is to determine non-specific gene reporter activation of compounds found active in a set of experiment entitled "Luminescence-based cell-based primary high throughput screening assay to identify activators of the DAF-12 from the parasite H. contortus (hcDAF-12)" (AID 652067). Compounds are tested in triplicate at a nominal concentration of 6.8 uM.

Protocol Summary:

In this assay, HEK293 are transfected with a GAL4-responsive reporter plasmid (tk-luc) and expression vectors encoding Gal4-LXR. The ability of compounds to alter LXR-mediated transcriptional activity is assessed by measuring luciferase expression from the reporter gene plasmid. The LXR agonist T0901317 was used as a positive control for this assay.

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 6.8 uM of T0901317 (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 3 uM Delta7 Dafachronic Acid
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:

The average percent inhibition and standard deviation of each compound tested were calculated. The sum of these two values was used as a hit cutoff parameter. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated 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 100-100, and for inactive compounds 90-0.

List of Reagents:

lit1-tk-luc luciferase reporter plasmid (Assay Provider)
hcDAF12 expressiong plasmid (Assay Provider)
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 11965)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
TransIT 293 (Mirus Corporation, part MIR-2700)
OptiMEM (Invitrogen, part 31985)
TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)
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. The MLSMR was not able to provide all compounds selected for testing in this assay.

External ID: SBCCG-A685-PEG3-Inh-Primary-Assay

Protocol: Assay Materials:
PEG-prom-Luc HeLa cells
DMEM 4.5g/l glu w/glu w/o phenol red
Fetal Bovine Serum (Hyclone)
L-glutamine (100X ) (Invitrogen)
Hygromycin B (CellGro)
Steady GLo (Promega)
Assay plate: Aurora 1536 white Solid Bottom Plate

I. Cell Suspension
1- Dispense 4 uL/well of assay media to columns 1-2 using Kalypsis dispenser.
2- Dispense 4 uL/well of cells at 3.75X10;5 cells/mL to columns 3-48 using Kalypsis Dispenser
3- Spin down plates on Eppendorf centrifuge 5810 at 500 rpm for 1 minute.

II. Compound Addition:
4- Using LabCyte Echo, transfer 40 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 40 nL of DMSO to control wells in Columns 1-4.
5- Spin down plates on Vspin at 1000 rpm for 1 minute.
6-Put Kalypsys metal lids on plates, and incubate plates at 37 degrees C with 5% CO2 overnight.

III. Reagent Addition
7- Retrieve plates from incubator, remove lids and allow to plates to cool at room temp for 10 minutes
8- Add 3uL/well of Steady-Glo reagent to all wells using Kalypsys.
9- Spin down plates without lids on Vspin at 2000 rpm for 2 min
10- Put lid on, and then incubate plates at room temp for 10 minutes.
IV. Reading plates:
11- Read the plate on PerkinElmer-EnVision plate reader luminescence protocol

Comment: Compounds that demonstrated % activity of >=50 at 20 uM concentration are defined as actives 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: CHEMBL4308277

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 21
First Page: 115089
Last Page: 115089
DOI: 10.1016/j.bmc.2019.115089

Target ChEMBL ID: CHEMBL613866
ChEMBL Target Name: CT26
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: CHEMBL4308276

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 21
First Page: 115089
Last Page: 115089
DOI: 10.1016/j.bmc.2019.115089

Target ChEMBL ID: CHEMBL613866
ChEMBL Target Name: CT26
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: CHEMBL4308275

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 21
First Page: 115089
Last Page: 115089
DOI: 10.1016/j.bmc.2019.115089

Target ChEMBL ID: CHEMBL394
ChEMBL Target Name: HCT-116
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: CHEMBL4308274

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 21
First Page: 115089
Last Page: 115089
DOI: 10.1016/j.bmc.2019.115089

Target ChEMBL ID: CHEMBL613866
ChEMBL Target Name: CT26
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: CHEMBL4308273

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 21
First Page: 115089
Last Page: 115089
DOI: 10.1016/j.bmc.2019.115089

Target ChEMBL ID: CHEMBL394
ChEMBL Target Name: HCT-116
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: CHEMBL4308272

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 21
First Page: 115089
Last Page: 115089
DOI: 10.1016/j.bmc.2019.115089

Target ChEMBL ID: CHEMBL614055
ChEMBL Target Name: SW48
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: CHEMBL4308271

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 21
First Page: 115089
Last Page: 115089
DOI: 10.1016/j.bmc.2019.115089

Target ChEMBL ID: CHEMBL394
ChEMBL Target Name: HCT-116
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: CHEMBL4308270

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 21
First Page: 115089
Last Page: 115089
DOI: 10.1016/j.bmc.2019.115089

Target ChEMBL ID: CHEMBL613866
ChEMBL Target Name: CT26
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: CHEMBL4308285

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 21
First Page: 115089
Last Page: 115089
DOI: 10.1016/j.bmc.2019.115089

Target ChEMBL ID: CHEMBL613866
ChEMBL Target Name: CT26
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: CHEMBL4308284

Protocol: N/A

Comment: Journal: Bioorg Med Chem
Year: 2019
Volume: 27
Issue: 21
First Page: 115089
Last Page: 115089
DOI: 10.1016/j.bmc.2019.115089

Target ChEMBL ID: CHEMBL613866
ChEMBL Target Name: CT26
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular