HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：N/A

External ID: CHEMBL4392930

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

Standard Type	Standard Relation	Standard Value
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：Chaperonin GroEL

External ID: CHEMBL4392931

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

Target ChEMBL ID: CHEMBL4296299
ChEMBL Target Name: 60 kDa chaperonin
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous single protein target assigned

PubChem Standard Value	Standard Type	Standard Relation	Standard Value	Standard Units	Data Validity Comment
250	IC50	>	250000	nM	Outside typical range
153	IC50	=	153000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
103	IC50	=	103000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range
250	IC50	>	250000	nM	Outside typical range

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：mu-type opioid receptor isoform MOR-1 [Homo sapiens]

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.

Activation at 9.3 uM
4.41
4.41
4.41
4.41
4.41
4.41
4.41
4.41
4.41
4.41
4.4
4.4
4.4
4.4
4.4
4.4
4.4
4.4
4.4
4.4

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：N/A

External ID: CHEMBL4392932

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

Standard Type	Standard Relation	Standard Value
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：disintegrin and metalloproteinase domain-containing protein 17 preproprotein [Homo sapiens]

External ID: ADAM17_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

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

Protocol Summary:

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

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

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

Where:

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

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

PubChem Activity Outcome and Score:

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

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

List of Reagents:

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

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

Inhibition at 6.95 uM
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23
1.23

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：10 kDa heat shock protein, mitochondrial

External ID: CHEMBL4392933

Protocol: N/A

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

Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

Target ChEMBL ID: CHEMBL4106131
ChEMBL Target Name: HSP60/HSP10
ChEMBL Target Type: PROTEIN COMPLEX - Target is a defined protein complex, consisting of multiple subunits
Relationship Type: D - Direct protein target assigned
Confidence: Direct protein complex subunits assigned

PubChem Standard Value	Standard Type	Standard Relation	Standard Value	Standard Units
20	IC50	=	20000	nM
1.3	IC50	=	1300	nM
2.8	IC50	=	2800	nM
100	IC50	>	100000	nM
3.2	IC50	=	3200	nM
100	IC50	>	100000	nM
2.1	IC50	=	2100	nM
4.5	IC50	=	4500	nM
15	IC50	=	15000	nM
12	IC50	=	12000	nM
100	IC50	>	100000	nM
11	IC50	=	11000	nM
17	IC50	=	17000	nM
2.8	IC50	=	2800	nM
16	IC50	=	16000	nM
100	IC50	>	100000	nM
6.3	IC50	=	6300	nM
2.3	IC50	=	2300	nM
100	IC50	>	100000	nM
23	IC50	=	23000	nM

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：Co-chaperonin GroES

External ID: CHEMBL4392934

Protocol: N/A

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

Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

Target ChEMBL ID: CHEMBL4106139
ChEMBL Target Name: GroEL/GroES
ChEMBL Target Type: PROTEIN COMPLEX - Target is a defined protein complex, consisting of multiple subunits
Relationship Type: D - Direct protein target assigned
Confidence: Direct protein complex subunits assigned

PubChem Standard Value	Standard Type	Standard Relation	Standard Value	Standard Units	Data Validity Comment
56	IC50	=	56000	nM
4.7	IC50	=	4700	nM
2	IC50	=	2000	nM
6.2	IC50	=	6200	nM
0.57	IC50	=	570	nM
178	IC50	=	178000	nM	Outside typical range
0.59	IC50	=	590	nM
18	IC50	=	18000	nM
2.5	IC50	=	2500	nM
0.49	IC50	=	490	nM
250	IC50	>	250000	nM	Outside typical range
5.2	IC50	=	5200	nM
66	IC50	=	66000	nM
13	IC50	=	13000	nM
30	IC50	=	30000	nM
2.4	IC50	=	2400	nM
0.29	IC50	=	290	nM
0.58	IC50	=	580	nM
250	IC50	>	250000	nM	Outside typical range
4	IC50	=	4000	nM

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：Co-chaperonin GroES

External ID: CHEMBL4392935

Protocol: N/A

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

Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

Target ChEMBL ID: CHEMBL4106139
ChEMBL Target Name: GroEL/GroES
ChEMBL Target Type: PROTEIN COMPLEX - Target is a defined protein complex, consisting of multiple subunits
Relationship Type: D - Direct protein target assigned
Confidence: Direct protein complex subunits assigned

PubChem Standard Value	Standard Type	Standard Relation	Standard Value	Standard Units
21	IC50	=	21000	nM
1.4	IC50	=	1400	nM
2.2	IC50	=	2200	nM
75	IC50	=	75000	nM
0.9	IC50	=	900	nM
100	IC50	>	100000	nM
1.3	IC50	=	1300	nM
7.3	IC50	=	7300	nM
9	IC50	=	9000	nM
6	IC50	=	6000	nM
100	IC50	>	100000	nM
8.5	IC50	=	8500	nM
24	IC50	=	24000	nM
4.3	IC50	=	4300	nM
13	IC50	=	13000	nM
100	IC50	>	100000	nM
3.8	IC50	=	3800	nM
0.36	IC50	=	360	nM
100	IC50	>	100000	nM
15	IC50	=	15000	nM

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：24565 靶标：N/A

External ID: ERK5 transcriptional activity-HTS

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

Comment:

Luciferase activity (AU)
104
108
64
28
100
152
176
52
124
44
60
60
32
96
60
144
28
64
84
44

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：muscarinic acetylcholine receptor M1 [Homo sapiens]

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.

Activation at 3 uM
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07
-1.07

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：N/A

External ID: CHEMBL4392936

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

Standard Type	Standard Relation	Standard Value
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：N/A

External ID: CHEMBL4392937

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

Standard Type	Standard Relation	Standard Value
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	>	10
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	=	1
Ratio IC50	>	10
Ratio IC50	=	1

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：Broad Institute 靶标：FATTY-ACID-CoA LIGASE FADD28 (FATTY-ACID-CoA SYNTHETASE)

External ID: 2147-01_Inhibitor_SinglePoint_HTS_Activity

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

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

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.

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

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

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

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

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

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

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

REPRODUCIBILITY_COSINE_TRANSFORM	REPLICATE_A_ACTIVITY_SCORE_12.48uM_(%)	REPLICATE_B_ACTIVITY_SCORE_12.48uM_(%)
0.9928	-1.516	-1.189
0.9728	-2.497	-1.536
0.9984	-3.184	-2.848
0.8083	-1.604	-0.252
0.1877	-1.639	1.113
0.8214	0.112	0.62
0.9999	-2.332	-2.278
0.9995	-3.841	-3.612
0.1219	-4.404	5.637
0.9897	-4.806	-3.591
0.9968	-8.098	-6.898
0.9517	-5.151	-2.639
0.7556	0.234	3.277
0.677	-0.034	0.82
0.9872	0.918	0.663
0.5086	-0.437	0.112
0.9056	-3.302	-1.195
0.0097	-1.83	1.795
0.9693	-0.764	-1.284
0.8976	-2.719	-0.928

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：Broad Institute 靶标：N/A

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

Protocol: Protocol

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

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

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

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

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

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

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

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

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

Comment:

REPRODUCIBILITY_COSINE_TRANSFORM	PCT_ACTIVE_REPLICATES	REPLICATE_A_ACTIVITY_SCORE_9.99uM_(%)
0	0	-208.81
0.9984	0	-216.092
0	0	-162.651
0	0	-162.095
0	0	-158.917
0	0	-156.183
0	0	-151.809
0	0	-151.967
0.9995	0	-161.183
0	0	-147.708
0.9989	0	-146.736
0	0	-141.126
0.9969	0	-165.263
0.9996	0	-146.86
1	0	-137.448
0.987	0	-136.764
0.999	0	-135.905
0	0	-134.44
0.9998	0	-139.162
0.9971	0	-155.151

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：N/A

External ID: CHEMBL4392928

Protocol: N/A

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

Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

PubChem Standard Value	Standard Type	Standard Relation	Standard Value	Standard Units
63	IC50	>	63000	nM
18	IC50	=	18000	nM
6.2	IC50	=	6200	nM
63	IC50	>	63000	nM
3	IC50	=	3000	nM
63	IC50	>	63000	nM
63	IC50	>	63000	nM
63	IC50	>	63000	nM
63	IC50	>	63000	nM
63	IC50	>	63000	nM
63	IC50	>	63000	nM
47	IC50	=	47000	nM
63	IC50	>	63000	nM
24	IC50	=	24000	nM
63	IC50	>	63000	nM
63	IC50	>	63000	nM
63	IC50	>	63000	nM
16	IC50	=	16000	nM
63	IC50	>	63000	nM
63	IC50	>	63000	nM

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：Thiosulfate sulfurtransferase

External ID: CHEMBL4392929

Protocol: N/A

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

Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

Target ChEMBL ID: CHEMBL4295835
ChEMBL Target Name: Thiosulfate sulfurtransferase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

PubChem Standard Value	Standard Type	Standard Relation	Standard Value	Standard Units
100	IC50	>	100000	nM
51	IC50	=	51000	nM
100	IC50	>	100000	nM
2.9	IC50	=	2900	nM
100	IC50	>	100000	nM
100	IC50	>	100000	nM
0.12	IC50	=	120	nM
100	IC50	>	100000	nM
1	IC50	=	1000	nM
0.28	IC50	=	280	nM
100	IC50	>	100000	nM
57	IC50	=	57000	nM
100	IC50	>	100000	nM
100	IC50	>	100000	nM
100	IC50	>	100000	nM
0.83	IC50	=	830	nM
0.06	IC50	=	60	nM
7.3	IC50	=	7300	nM
100	IC50	>	100000	nM
2.3	IC50	=	2300	nM

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：muscarinic acetylcholine receptor M1 [Homo sapiens]

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.

Activation at 3 uM
9.75
9.75
9.75
9.75
9.75
9.75
9.75
9.75
9.75
9.75
9.75
9.75
9.75
9.74
9.74
9.74
9.74
9.74
9.74
9.74

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：RecName: Full=Sialate O-acetylesterase; AltName: Full=H-Lse; AltName: Full=Sialic acid-specific 9-O-acetylesterase; Flags: Precursor [Homo sapiens]

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.

Inhibition at 9.66 uM
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91
6.91

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：

External ID: epac1-activator-f1f2f3f4

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

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

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

Phenotype	Fit_LogAC50	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.00260 uM	Activity at 0.00420 uM	Activity at 0.00779 uM	Activity at 0.023 uM	Activity at 0.070 uM	Activity at 0.148 uM	Activity at 0.213 uM	Activity at 0.637 uM	Activity at 1.910 uM	Activity at 3.487 uM	Activity at 5.719 uM	Activity at 17.11 uM	Activity at 51.33 uM	Activity at 83.48 uM	Activity at 153.7 uM	Compound QC
Inactive	-4.2	1	0.8542	15	-0.8141	4	0 0 0 0 0 0 0 0	12.4761		0.6149		0.1958		-4.8451		1.0169		-0.1665		0.9292		7.324	12.4761	QC'd by Chem Div
Inactive				0	0	4		5.5368						-0.1521		-0.4853		0.8123		3.04		4.3508	5.5368	QC'd by Chem Div
Inactive				0	0	4		8.6042		4.8618		6.0005		4.8572		2.4088		2.5501		1.8735		3.3336	8.6042	QC'd by Chem Div
Inactive				0	0	4		-2.8803		1.5268		-0.8493		-0.8611		4.1068		-1.778		-0.0897		-1.4954	-2.8803	QC'd by Chem Div
Inactive				0	0	4		6.6979		5.8514		4.5387		3.2655		6.7193		3.1377		3.8268		4.8314	6.6979	QC'd by Chem Div
Inactive				0	0	4		11.2642						3.9196		1.8947		4.7539		2.7499		4.3951	11.2642	QC'd by Chem Div
Inactive				0	0	4		4.3429						2.9262		-0.1885		3.2488		1.5027		-2.5002	4.3429	QC'd by Chem Div
Inactive				0	0	4		-0.6829						3.3556		1.3973		2.4698		3.7138		2.6913	-0.6829	QC'd by DPISMR
Inactive				0	0	4		4.1094		2.978		2.9594		-0.2137		3.9629		3.2444		7.0105		2.2449	4.1094	QC'd by ChemBridge
Inactive	-3.9	1	0.7748	30	0.6852	4	0 0 0 0 0 0 0 0	23.3109		4.9223		1.4858		-0.2624		1.7621		-0.0889		1.7568		6.6429	23.3109	QC'd by ChemBridge
Inactive				0	0	4		7.2259						1.1257		2.5045		2.118		2.136		0.3108	7.2259	QC'd by DPISMR
Inactive				0	0	4		2.7681		6.7634		2.2278		3.3419		0.9846		1.3115		2.7671		3.3811	2.7681	QC'd by ChemBridge
Inactive	-4.1	1	0.8429	18	-0.1632	4	0 0 0 0 0 0 0 0	13.8334		2.7619		1.5007		-1.4428		1.3099		-2.636		1.8618		7.5982	13.8334	QC'd by ChemBridge
Inactive				0	0	4		5.8128						-3.6075		1.4591		2.0274		-0.4652		-4.916	5.8128	QC'd by ChemBridge
Inactive	-3.95	1	0.7398	-12.738	-0.5	4	0 0 0 0 0 0	-8.9483						-1.7034		-0.1105		-2.1462		1.7984		-5.297	-8.9483	QC'd by ChemBridge
Inactive				0	0	4		-2.5811		-8.353		-2.4919		-8.4727		-4.4155		-5.0949		-1.5077		-0.4001	-2.5811	QC'd by ChemBridge
Inactive	-4.45	1	0.7869	-2	9	4	0 0 0 0 0 0 0 0	0.1751		11.5214		7.293		5.7774		10.9823		7.558		6.13		1.2752	0.1751	QC'd by ChemBridge
Inactive	-3.95	1	0.8443	24	-0.6883	4	0 0 0 0 0 0 0 0	17.1313		-1.3595		1.4567		-3.4902		0.3518		2.6702		1.9371		6.158	17.1313	QC'd by ChemBridge
Inactive	-6.65	1	0.4363	3	-6.188	4	0 0 0 0 0 0	7.2613						-3.49		1.1499		3.8934		1.143		-1.0367	7.2613	QC'd by ChemBridge
Inactive		0.6	0.956	-7.3878	7.5	4	0 0 0 0 0 0 0 0 0 0 0	-7.4065	6.3748		5.7815	8.5267	5.2031		4.2367	3.0265	-1.7308		-2.2978	-3.391	-4.7307		-7.4065	QC'd by Microsource

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：

External ID: epac1-inhibitor-f1f2f3f4

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

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

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

Phenotype	Potency	Efficacy	Activity_Score	Curve_Description	Fit_LogAC50	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.00194 uM	Activity at 0.00314 uM	Activity at 0.00582 uM	Activity at 0.017 uM	Activity at 0.052 uM	Activity at 0.111 uM	Activity at 0.159 uM	Activity at 0.476 uM	Activity at 1.427 uM	Activity at 2.605 uM	Activity at 4.273 uM	Activity at 12.78 uM	Activity at 38.35 uM	Activity at 62.38 uM	Activity at 114.8 uM	Compound QC
Inhibitor	3.1414	91.5653	84	Complete curve; high efficacy	-5.5029	1.1	0.996	-83.5653	8	-1.1	0 0 0 0 0 0 0 0 0 0 0	-83.3985	5.8642		5.1752	6.8922	10.9031		5.3804	2.223	-20.9811		-45.6671	-65.9774	-77.4953		-83.3985	QC'd by SigmaAldrich
Inhibitor	7.5686	73.193	83	Complete curve; high efficacy	-5.121	1.7885	0.9952	-82.8887	-9.6957	-1.1	0 0 0 0 0 0 0 0	-80.375		-9.2369		-7.6804		-7.6509		-14.8323		-17.372		-58.9614		-82.7971	-80.375	QC'd by Chem Div
Inhibitor	9.5283	113.417	83	Complete curve; high efficacy	-5.021	2.0479	0.9957	-126.8199	-13.4029	-1.1	0 0 0 0 0 0 0 0	-128.8821		-12.2785		-9.679		-19.3504		-12.0769		-19.5794		-82.8273		-119.9921	-128.8821	QC'd by Asinex Ltd.
Inhibitor	8.9125	97.0941	83	Complete curve; high efficacy	-5.05	1.4163	0.9972	-95.9137	1.1804	-1.1	0 0 0 0 0 0	-95.1525						-2.2262		3.0431		-10.9978		-55.3438		-87.9728	-95.1525	QC'd by Key Organics Ltd.
Inhibitor	7.5686	80.842	83	Complete curve; high efficacy	-5.121	1.8617	0.99	-94.8681	-14.0261	-1.1	0 0 0 0 0 0 0 0	-88.9598		-9.2218		-14.3086		-16.5014		-16.2031		-22.3172		-68.1032		-98.8209	-88.9598	QC'd by Sigma Chemical Company
Inhibitor	9.5283	107.836	83	Complete curve; high efficacy	-5.021	0.8	0.9912	-121.2518	-13.4158	-1.1	0 0 0 0 0 0 0 0	-109.8296		-16.8462		-9.939		-19.9812		-16.655		-43.876		-69.7913		-99.6793	-109.8296	QC'd by Asinex Ltd.
Inhibitor	9.5283	102.4102	83	Complete curve; high efficacy	-5.021	2.2526	0.9928	-112.0345	-9.6243	-1.1	0 0 0 0 0 0 0 0	-116.7026		-13.4215		-5.2335		-14.5215		-9.9722		-10.6725		-70.1375		-106.0787	-116.7026	QC'd by InterBioScreen
Inhibitor	6.7456	100.7477	83	Complete curve; high efficacy	-5.171	2.0479	0.9883	-102.4434	-1.6957	-1.1	0 0 0 0 0 0 0 0	-110.1541		-0.6199		-3.1798		-6.2849		2.8918		-11.5064		-77.2529		-92.783	-110.1541	QC'd by InterBioScreen
Inhibitor	8.4921	112.572	83	Complete curve; high efficacy	-5.071	2.0479	0.9978	-114.9669	-2.3949	-1.1	0 0 0 0 0 0 0 0	-112.6964		-0.4977		-0.1079		-6.8481		-0.5406		-11.4336		-76.2713		-113.3796	-112.6964	QC'd by Chem Div
Inhibitor	9.5283	100.6484	83	Complete curve; high efficacy	-5.021	1.7885	0.9962	-100.6484	0	-1.1	0 0 0 0 0 0 0 0	-102.9125		3.2274		0.2085		-3.8063		-2.2579		-5.9804		-58.4009		-93.2587	-102.9125	QC'd by InterBioScreen
Inhibitor	7.0327	75.8075	82	Complete curve; high efficacy	-5.1529	1.21	0.9887	-67.8075	8	-1.1	0 0 0 0 0 0 0 0 0 0 0	-62.7329	6.4136		9.158	7.5581	6.5704		5.8253	3.2639	4.0875		-18.7337	-46.8788	-54.1698		-62.7329	QC'd by Timtec
Inhibitor	9.5283	77.6809	82	Complete curve; high efficacy	-5.021	1.2221	0.9944	-83.7222	-6.0413	-1.1	0 0 0 0 0 0 0 0	-81.7738		-4.3587		-3.5127		-10.8793		-7.6754		-16.5948		-51.1312		-73.833	-81.7738	QC'd by InterBioScreen
Inhibitor	10.691	75.7058	82	Complete curve; high efficacy	-4.971	1.9673	0.9723	-81.5585	-5.8527	-1.1	0 0 0 0 0 0 0 0	-71.231		-3.7858		-1.8418		-9.4119		-9.4119		-9.0819		-46.725		-88.2668	-71.231	QC'd by Chem Div
Inhibitor	6.012	99.4255	82	Complete curve; high efficacy	-5.221	0.4	0.9552	-66.4076	33.018	-1.1	0 0 0 0 0 0 0 0	-48.5497		32.9302		23.8568		11.216		1.8455		-3.1495		-12.7102		-42.9961	-48.5497	QC'd by InterBioScreen
Inhibitor	12.5893	84.4035	82	Complete curve; high efficacy	-4.9	2.7202	0.9858	-86.2417	-1.8382	-1.1	0 0 0 0 0 0	-85.684						2.3129		-10.9395		1.6385		-38.1285		-86.0696	-85.684	QC'd by Nathanael S Gray - Dana Farber Cancer Institute - MLI PSL
Inhibitor	12.5893	81.6146	82	Complete curve; high efficacy	-4.9	2.3332	0.999	-91.3016	-9.687	-1.1	0 0 0 0 0 0	-91.1194						-8.8096		-11.8548		-10.1481		-45.0265		-88.9937	-91.1194	QC'd by InterBioScreen
Inhibitor	13.4591	86.9588	82	Complete curve; high efficacy	-4.871	1.4641	0.9849	-93.9971	-7.0383	-1.1	0 0 0 0 0 0 0 0	-84.5472		-4.3953		-7.2317		-9.2129		-14.3512		-7.0393		-45.0179		-87.0343	-84.5472	QC'd by InterBioScreen
Inhibitor	8.9125	91.1397	82	Complete curve; high efficacy	-5.05	4.095	0.9946	-89.0564	2.0833	-1.1	0 0 0 0 0 0	-93.3505						5.8165		-1.4499		2.1549		-65.2799		-85.2894	-93.3505	QC'd by Enamine
Inhibitor	15.1014	90.2598	82	Complete curve; high efficacy	-4.821	2.4064	0.9711	-110.2608	-20.001	-1.1	0 0 0 0 0 0 0 0	-98.3869		-17.9349		-21.0527		-21.4562		-23.8802		-18.187		-46.7884		-119.3299	-98.3869	QC'd by NCI Chemotherapeutic Agents Repository
Inhibitor	15.7441	90.0908	81	Complete curve; high efficacy	-4.8029	1.6604	0.9839	-89.1483	0.9425	-1.1	0 0 0 0 0 0 0 0 0 0 0	-83.9438	2.967		7.2054	2.7371	-0.6122		2.788	-2.9535	-4.3083		-13.5377	-29.9354	-76.8118		-83.9438	QC'd by SigmaAldrich

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：

External ID: epac1-activator-v

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

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

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

Phenotype	Potency	Efficacy	Activity_Score	Curve_Description	Fit_LogAC50	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.025 uM	Activity at 0.120 uM	Activity at 0.611 uM	Activity at 3.058 uM	Activity at 15.29 uM	Activity at 75.76 uM	Activity at 149.6 uM	Compound QC
Activator	112.2018	2659.1944	100	Partial curve; high efficacy	-3.95	4.5045	0.9988	2689.24	30.0456	2.1	0 0 0 0 0 0	2175.7605		-2.1864	18.441	20.7973	83.3849	426.7958	2175.7605	QC'd by Microsource
Activator	0.1778	128.3128	95	Complete curve; high efficacy	-6.75	4.9549	0.9927	134.4851	6.1723	1.1	0 0 0 0 0 1	14.509	5.6033	24.7938	142.7655	131.831	127.7753	14.509		QC'd by SigmaAldrich
Activator	0.4467	126.1077	91	Complete curve; high efficacy	-6.35	1.8617	0.9998	123.6363	-2.4714	1.1	0 0 0 0 0 1	0.2688	-2.5844	7.8161	80.257	120.2764	123.3895	0.2688		QC'd by SigmaAldrich
Activator	1.122	117.1254	90	Complete curve; high efficacy	-5.95	2.2481	0.9788	132.4265	15.3011	1.1	0 0 0 0 0	143.3187		15.8094	39.5652	122.9227	119.7824	143.3187		QC'd by Tocris
Activator	112.2018	1122.1776	71	Partial curve; high efficacy	-3.95	4.5045	0.9983	1140.5688	18.3912	2.1	0 0 0 0 0	922.7903			3.2305	7.5889	41.6506	204.8302	922.7903	QC'd by Timtec
Activator	100	709.5673	58	Partial curve; high efficacy	-4	3.99	0.9988	700.7079	-8.8594	2.1	0 0 0 0 0 0	592.8155		-15.9019	-8.6532	4.5286	-20.153	176.4838	592.8155	QC'd by CarsonNewman-SPECS
Activator	79.4328	826.4076	57	Partial curve; high efficacy	-4.1	4.095	0.9972	826.7667	0.3591	2.1	0 0 0 0 0 0	665.6736		-2.5729	-3.4508	-16.7617	25.5851	154.5978	665.6736	QC'd by Prestwick Chemical; Inc.
Activator	100	589.9325	55	Partial curve; high efficacy	-4	3.2475	0.984	613.2979	23.3654	2.1	0 0 0 0 0 0	498.6162		-5.747	1.059	37.6543	62.5313	194.9599	498.6162	QC'd by CarsonNewman-SPECS
Activator	112.2018	558.6566	55	Partial curve; high efficacy	-3.95	4.5045	0.9992	557.2619	-1.3947	2.1	0 0 0 0 0	448.681			-6.8565	-3.8767	7.0632	81.1858	448.681	QC'd by Vitas
Activator	100	612.4753	55	Partial curve; high efficacy	-4	4.5045	0.9972	611.2868	-1.1885	2.1	0 0 0 0 0 0	533.4091		-10.048	-5.6002	-8.2068	21.4265	142.7223	533.4091	QC'd by Pharmacopeia
Activator	100	511.5518	53	Partial curve; high efficacy	-4	4.4495	0.9965	517.5467	5.995	2.1	0 0 0 0 0 0	453.9884		-10.4867	10.4448	3.9744	21.1983	121.4017	453.9884	QC'd by CarsonNewman-SPECS
Activator	112.2018	440.1981	52	Partial curve; high efficacy	-3.95	4.095	0.9989	439.2753	-0.9228	2.1	0 0 0 0 0	348.6312			-5.1918	-3.3018	6.9705	74.6909	348.6312	QC'd by Sequoia
Activator	100	497.635	52	Partial curve; high efficacy	-4	3.6772	0.9977	486.8252	-10.8098	2.1	0 0 0 0 0 0	401.671		-14.9427	-21.3461	-13.1864	2.6858	130.5049	401.671	QC'd by Pharmacopeia
Activator	100	438.5085	51	Partial curve; high efficacy	-4	4.9549	0.9987	437.7956	-0.7128	2.1	0 0 0 0 0 0	392.29		-7.2531	0.2913	-4.176	9.3666	93.145	392.29	QC'd by Pharmacopeia
Activator	56.2341	819.9718	50	Partial curve; high efficacy	-4.25	3.2475	0.9993	802.9227	-17.049	2.1	0 0 0 0 0	787.1792			-7.4836	-10.9833	-14.2075	580.1833	787.1792	QC'd by NCI
Activator	112.2018	363.641	50	Partial curve; high efficacy	-3.95	4.9549	0.9972	355.9752	-7.6658	2.1	0 0 0 0 0 0	293.7089		-16.7363	0.4741	-6.1211	-4.3689	34.2599	293.7089	QC'd by Pharmacopeia
Activator	112.2018	363.0297	50	Partial curve; high efficacy	-3.95	4.5045	0.9992	370.4299	7.4002	2.1	0 0 0 0 0 0	299.7006		3.6943	4.7004	13.0282	5.8575	63.7543	299.7006	QC'd by Pharmacopeia
Activator	100	295.8134	48	Partial curve; high efficacy	-4	3.6272	0.9992	302.0389	6.2255	2.1	0 0 0 0 0 0	251.6991		2.6798	8.4811	8.6778	9.6504	86.3825	251.6991	QC'd by CarsonNewman-SPECS
Activator	100	233.3469	46	Partial curve; high efficacy	-4	4.5045	0.9961	221.7227	-11.6242	2.1	0 0 0 0 0 0	192.4676		-6.6471	-16.5394	-18.4762	-5.6031	42.3638	192.4676	QC'd by CarsonNewman-SPECS
Activator	112.2018	234.6061	46	Partial curve; high efficacy	-3.95	4.9549	0.984	235.2356	0.6295	2.1	0 0 0 0 0 0	197.0148		0.9163	-4.5136	1.9482	17.442	18.7867	197.0148	QC'd by Pharmacopeia

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：Burnham Center for Chemical Genomics 靶标：N/A

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

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

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

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

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

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

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

%Activity at 25 uM	Value at 25uM	Mean High	STD Deviation High	Mean Low	STD Deviation Low
-0.26	39.359	49.23	0.68	39.39	0.67
3.98	39.777	49.23	0.68	39.39	0.67
-0.26	39.359	49.23	0.68	39.39	0.67
1.15	39.498	49.23	0.68	39.39	0.67
3.98	39.777	49.23	0.68	39.39	0.67
2.56	39.637	49.23	0.68	39.39	0.67
6.8	40.055	49.23	0.68	39.39	0.67
5.39	39.916	49.23	0.68	39.39	0.67
5.39	39.916	49.23	0.68	39.39	0.67
1.15	39.498	49.23	0.68	39.39	0.67
1.15	39.498	49.23	0.68	39.39	0.67
-0.26	39.359	49.23	0.68	39.39	0.67
-1.68	39.22	49.23	0.68	39.39	0.67
2.56	39.637	49.23	0.68	39.39	0.67
5.39	39.916	49.23	0.68	39.39	0.67
2.56	39.637	49.23	0.68	39.39	0.67
3.98	39.777	49.23	0.68	39.39	0.67
-1.68	39.22	49.23	0.68	39.39	0.67
2.56	39.637	49.23	0.68	39.39	0.67
2.56	39.637	49.23	0.68	39.39	0.67

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：

External ID: epac1-inhibitor-v

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

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

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

Phenotype	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.120 uM	Activity at 0.202 uM	Activity at 0.611 uM	Activity at 1.089 uM	Activity at 3.058 uM	Activity at 5.503 uM	Activity at 15.29 uM	Activity at 27.41 uM	Activity at 75.76 uM	Activity at 149.6 uM	Activity at 201.4 uM	Compound QC
Inactive	4.9549	0.7824	1.5059	12.5	4	0 0 0 0 0 0	2.5014	8.6579		16.7333		7.679		-0.4118		3.2398	2.5014		QC'd by Pharmacopeia
Inactive	4.9549	0.5059	2	22.5	4	0 0 0 0 0 0	6.2536	23.1671		31.5741		20.3971		12.3231		23.6931	6.2536		QC'd by Pharmacopeia
Inactive	3.0654	0.4875	6	14	4	0 0 0 0 0 0	7.1178	13.8537		13.0635		10.2396		19.9104		9.8496	7.1178		QC'd by Pharmacopeia
Inactive	4.9549	0.8513	-6.1222	14.5	4	0 0 0 0 0 0	-2.6019	18.6181		15.6971		11.9589		10.6497		12.3116	-2.6019		QC'd by Pharmacopeia
Inactive	4.9549	0.444	1	-8.9353	4	0 0 0 0 0 0	4.128	-6.6128		2.8591		5.135		-4.2457		-1.6866	4.128		QC'd by Pharmacopeia
Inactive	0.9	0.6078	1.5	7	4	0 0 0 0 0 0	3.2802	4.9355		7.5212		2.2467		1.4536		0.7626	3.2802		QC'd by Pharmacopeia
Inactive					4		15.8078	23.8467		15.2102		5.4007		17.1177		11.7038	15.8078		QC'd by Pharmacopeia
Inactive					4		-4.3249	4.3564		0.9262		-8.2609		6.6136		3.2318	-4.3249		QC'd by Pharmacopeia
Inactive	4.9549	0.386	6.5	16	4	0 0 0 0 0 1	18.5613	14.4696		22.6167		10.2425		16.2585		8.0741	18.5613		QC'd by Pharmacopeia
Inactive	4.9549	0.7935	15	-5.3851	4	0 0 0 0 0 0	16.1015	-1.1543		20.9641		12.4561		14.2395		11.4227	16.1015		QC'd by Pharmacopeia
Inactive	4.5045	0.9473	10.5	22	4	0 0 0 0 0 0	8.6846	21.3965		22.3063		12.7817		10.4065		12.724	8.6846		QC'd by Pharmacopeia
Inactive	0.9	0.7228	2.6804	26	4	0 0 0 0 0 0	-2.7663	19.5819		6.4245		5.729		8.0359		4.8741	-2.7663		QC'd by Pharmacopeia
Inactive	4.4495	0.861	16.5	8.5	4	0 0 0 0 0 1	9.315	8.5106		10.8538		6.2271		15.3684		16.5398	9.315		QC'd by Pharmacopeia
Inactive	4.9549	0.5741	10.5	0.7083	4	0 0 0 0 0 0	12.2191	4.1249		-2.3265		14.4634		3.8399		10.7866	12.2191		QC'd by Pharmacopeia
Inactive					4		9.7084	13.4221		8.3748		0.3338		10.1018		18.0457	9.7084		QC'd by Pharmacopeia
Inactive	4.9549	0.4502	-7.4141	8.5	4	0 0 0 0 0 0	-4.5118	9.0559		14.9898		-2.0146		10.2874		8.4395	-4.5118		QC'd by Pharmacopeia
Inactive	4.9549	0.6758	28	0.2443	4	0 0 0 0 0 0	23.0495	7.0826		-6.4631		4.1349		2.386		-6.0302	23.0495		QC'd by Pharmacopeia
Inactive	4.4495	0.7608	-2.5925	6.5	4	0 0 0 0 0 1	8.9695	3.4459		5.6768		9.8855		-0.0456		-2.1604	8.9695		QC'd by Pharmacopeia
Inactive	2.6384	0.665	13.5	20	4	0 0 0 0 0 0	13.5742	16.0359		22.5098		21.2723		19.3862		14.3408	13.5742		QC'd by Pharmacopeia
Inactive					4		-3.2632		-16.2622		-13.7616		-11.6684		-9.5243		-17.6211	-3.2632	QC'd by Prestwick Chemical; Inc.

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：15378 靶标：N/A

External ID: TELO_02

Protocol: HCT116 colorectal cancer cells are infected with Ad-hTR-mut and cytotoxicity is assessed 5-days post infection, measured by standard MTT assay. Ad-hTR-mut constructed using the Adeasy system (Agilent) and purified and quantified using Adeno-x purification and rapid titre kits (Clontech). Cells are seeded at 10000 cells per well and infected with 500pfu/cell in 96 well plates (Costar) then incubated for 48h prior to addition of compounds. Compounds were screened at 10uM in duplicate 2 days post-infection and MTT assay performed 3 days later at 5 days post-infection. A negative control adenovirus was included on every plate as a control for non-specific viral toxicity and all wells were expressed relative to untreated cells. Absorbance at 570nm was determined using a Molecular Devices spectramax plus instrument and analysed using SoftMax Pro software. Hits were taken to be compounds giving greater than 2.5-fold increase in absorbance indicating increased cell survival.

Comment: Target cell viability at time of assay was 30-50% after infection with this stock of Ad-hTR-mut. Compounds giving 2.5-fold increase in survival relative to this condition were taken to be hits.

PUBCHEM_CID	Average fold change at 10uM
2835245	2.37345151761089
5762732	2.36991761817046
273724	2.3633566237567
428044	2.36007795439464
4372721	2.35604391203318
3981587	2.35272774293471
5761810	2.33290828855493
2829606	2.31292518175006
5866141	2.30539893051177
2931161	2.27513175470908
1040370	2.27281433832202
2814240	2.26165663543268
6373007	2.25090778161813
673234	2.24926143067941
5190801	2.2419406617287
2835207	2.22660017924689
5280960	2.21063591324776
4655971	2.19507356532407
170475	2.19458458305062
2827343	2.18358659086153

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：

External ID: epac2-inhibitor-f1f2f3f4

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

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

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

Phenotype	Potency	Efficacy	Activity_Score	Curve_Description	Fit_LogAC50	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.00193 uM	Activity at 0.00314 uM	Activity at 0.00579 uM	Activity at 0.017 uM	Activity at 0.052 uM	Activity at 0.110 uM	Activity at 0.158 uM	Activity at 0.473 uM	Activity at 1.419 uM	Activity at 2.593 uM	Activity at 4.249 uM	Activity at 12.71 uM	Activity at 38.14 uM	Activity at 62.54 uM	Activity at 114.2 uM	Compound QC
Inhibitor	6.7456	1494.3646	100	Complete curve; high efficacy	-5.171	2.6384	0.965	-1503.2555	-8.8908	-1.1	0 0 0 0 0 0 0 0	-1260.1318		-11.1547		-6.7992		-1.6069		13.7817		-128.8233		-1191.5094		-1764.3843	-1260.1318	QC'd by Enamine
Inhibitor	0.0953	1682.333	92	Complete curve; high efficacy	-7.021	1.4787	0.9805	-97.0681	1585.265	-1.1	1 0 0 0 0 0 0 0	-38.6964		3173.9085		1467.8379		734.0242		61.6921		-105.8029		-252.9709		21.2579	-38.6964	QC'd by Richard A Houghten - Torrey Pines Institute for Molecular Studies - MLI PSL
Inhibitor	0.9528	462.0855	91	Complete curve; high efficacy	-6.021	4.9549	0.8781	-147.1919	314.8936	-1.1	1 0 1 0 0 0 0 0	-170.5569		526.5921		229.1516		847.3074		389.4244		-222.0195		-22.4759		-166.4298	-170.5569	QC'd by Chem Div
Inhibitor	0.0675	24.591	86	Complete curve; high efficacy	-7.171	3.1925	0.4734	-42.2643	-66.8553	-1.1	0 0 0 0 0 0 0 1	-61.4189		-67.8557		-65.4209		-48.882		-33.4136		-32.3036		-35.0315		-67.9216	-61.4189	QC'd by Scott E Schaus - Boston Univ. - MLI CMLD
Inhibitor	0.019	21.6073	86	Complete curve; high efficacy	-7.721	3.0654	0.8497	-38.71	-60.3172	-1.1	0 0 0 0 0 0	-33.8968		-60.016		-50.4881		-38.7102		-36.9245		-45.4763		-33.8968				QC'd by Specs
Inhibitor	5.548	138.1821	86	Complete curve; high efficacy	-5.2559	1.3723	0.995	-149.829	-11.6469	-1.1	0 0 0 0 0 0 0 0 0 0 0	-146.0322	-15.3641		-14.426	-10.6845	-2.5307		-13.6678	-14.3878	-34.5896		-65.4595	-119.9073	-140.2727		-146.0322	QC'd by Tocris
Inhibitor	1.1995	6.0318	86	Complete curve; high efficacy	-5.921	4.9549	0.4811	-77.4305	-71.3987	-1.1	0 0 0 0 0 0 0 1	-69.1964		-75.7075		-69.7865		-73.9043		-66.5822		-78.692		-74.9799		-77.8985	-69.1964	QC'd by NIH Chemical Genomics Center (NCGC)
Inhibitor	3.0131	74.5196	85	Complete curve; high efficacy	-5.521	1.2475	0.976	-90.5995	-16.08	-1.1	0 0 0 0 0 0 0 0	-88.471		-25.2771		-12.4556		-14.1585		-17.913		-51.4226		-73.9001		-93.7883	-88.471	QC'd by Key Organics Ltd.
Inhibitor	1.6944	72.7439	85	Complete curve; high efficacy	-5.771	1.6266	0.9736	-86.1149	-13.3709	-1.1	0 0 0 0 0 0 0 0	-90.2672		-16.7468		-16.0693		-11.6766		-14.4415		-64.3096		-72.7308		-87.9452	-90.2672	QC'd by Asinex Ltd.
Inhibitor	0.151	140.6456	85	Complete curve; high efficacy	-6.821	0.9	0.9849	-44.2164	-184.862	-1.1	0 0 0 0 0 0	-53.3465		-180.1774		-166.0242		-127.2248		-89.878		-43.6723		-53.3465				QC'd by Sytravon
Inhibitor	2.4782	71.9197	85	Complete curve; high efficacy	-5.6059	1.21	0.9515	-90.347	-18.4273	-1.1	0 0 0 0 0 0 0 0 0 0 0	-92.3794	-29.4176		-25.4576	-10.6267	-18.3361		-13.1226	-18.0422	-51.5862		-63.4387	-81.7748	-84.6589		-92.3794	QC'd by Microsource
Inhibitor	2.6855	76.2541	85	Complete curve; high efficacy	-5.571	1.5095	0.9645	-93.4891	-17.235	-1.1	0 0 0 0 0 0 0 0	-83.3595		-19.1979		-15.5958		-20.1946		-17.5552		-53.8059		-81.8551		-105.9967	-83.3595	QC'd by Specs
Inhibitor	3.0131	93.5998	85	Complete curve; high efficacy	-5.521	4.095	0.9841	-106.6274	-13.0276	-1.1	0 0 0 0 0 0 0 0	-100.9847		-15.8586		-12.6799		-7.6231		-13.2588		-38.4269		-100.335		-118.4749	-100.9847	QC'd by Chem Div
Inhibitor	2.2087	70.7868	85	Complete curve; high efficacy	-5.6559	1.4641	0.9882	-83.9058	-13.119	-1.1	0 0 0 0 0 0 0 0 0 0 0	-80.3383	-14.6034		-13.7105	-8.1697	-17.0327		-13.9823	-20.0222	-32.5347		-69.34	-76.772	-86.3228		-80.3383	QC'd by Prestwick Chemical; Inc.
Inhibitor	16.9441	355.3528	85	Complete curve; high efficacy	-4.771	1.1	0.9993	-356.9435	-1.5907	-1.1	0 0 0 0 0 0 0 0	-318.1315		-3.9948		-2.9251		-4.5312		0		-40.0216		-141.0144		-284.0357	-318.1315	QC'd by Enamine
Inhibitor	5.3582	75.6887	84	Complete curve; high efficacy	-5.271	1.1	0.986	-94.241	-18.5523	-1.1	0 0 0 0 0 0 0 0	-92.9398		-25.1237		-17.5814		-17.053		-18.0751		-44.0369		-68.5795		-88.598	-92.9398	QC'd by Jeff Aube - Univ. of Kansas - MLI CMLD
Inhibitor	0.7569	135.5837	84	Complete curve; high efficacy	-6.121	1.3437	0.7835	-54.4989	81.0848	-1.1	1 0 0 0 1 0 0 0	0		114.0331		82.5711		72.5433		33.2174		-170.3018		-49.8526		-108.2167	0	QC'd by Sergey Kozmin - Univ. of Chicago - MLI CMLD
Inhibitor	4.7755	97.0719	84	Complete curve; high efficacy	-5.321	0.8	0.9918	-103.7002	-6.6283	-1.1	0 0 0 0 0 0 0 0	-94.1135		-1.4836		-13.9984		-10.0011		-18.7058		-41.6061		-71.5982		-94.9636	-94.1135	QC'd by Key Organics Ltd.
Inhibitor	3.7933	76.1919	84	Complete curve; high efficacy	-5.421	1.1	0.9724	-84.3357	-8.1438	-1.1	0 0 0 0 0 0 0 0	-80.1829		0.3249		-17.896		-10.6249		-19.5352		-32.7938		-69.8932		-81.1131	-80.1829	QC'd by ChemBridge
Inhibitor	3.0131	69.4925	84	Complete curve; high efficacy	-5.521	1.5579	0.9812	-74.2039	-4.7114	-1.1	0 0 0 0 0 0 0 0	-65.987		-5.0213		-5.2016		-3.1865		-8.3924		-32.9126		-65.2042		-82.0627	-65.987	QC'd by Asinex Ltd.

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：

External ID: epac2-activator-f1f2f3f4

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

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

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

Phenotype	Potency	Efficacy	Activity_Score	Curve_Description	Fit_LogAC50	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.00260 uM	Activity at 0.00423 uM	Activity at 0.00779 uM	Activity at 0.023 uM	Activity at 0.070 uM	Activity at 0.148 uM	Activity at 0.213 uM	Activity at 0.637 uM	Activity at 1.910 uM	Activity at 3.487 uM	Activity at 5.719 uM	Activity at 17.11 uM	Activity at 51.33 uM	Activity at 83.79 uM	Activity at 153.7 uM	Compound QC
Activator	112.2018	7915.679	100	Partial curve; partial efficacy	-3.95	4.9549	0.974	7782.4016	-133.2774	2.2	0 0 0 0 0 0 0 0	6550.8431		5.0383		1.4327		-1.8529		-0.749		-1.2828		-0.8048		-0.2208	6550.8431	QC'd by Asinex Ltd.
Activator	112.2018	19185.7415	100	Partial curve; partial efficacy	-3.95	4.9549	0.9741	18856.4676	-329.2738	2.2	0 0 0 0 0 0 0 0	15872.4475		-8.7565		-6.4408		0.7001		-5.8905		-4.8567		-9.6925		-5.1865	15872.4475	QC'd by UrkORgSynthesis Ltd
Activator	112.2018	2376.3081	100	Partial curve; partial efficacy	-3.95	4.5045	0.9633	2450.5257	74.2175	2.2	0 0 0 0 0 0 0 0	1992.2973		-3.5317		-4.7193		54.5184		28.6317		18.0366		392.2696		419.7454	1992.2973	QC'd by ChemBridge
Activator	100	5725.9134	100	Partial curve; partial efficacy	-4	4.045	0.9954	5798.942	73.0286	2.2	0 0 0 0 0 0 0 0	4931.0731		0.8803		0.5649		-0.7911		3.8185		42.4751		348.6751		1565.7548	4931.0731	QC'd by NCI Chemotherapeutic Agents Repository
Activator	15.8489	881.2232	93	Complete curve; high efficacy	-4.8	2.2526	0.9634	878.8998	-2.3234	1.1	0 0 0 0 0 0 0 0	728.8831		-4.2226		-4.7068		-6.1137		-1.0929		20.5614		426.6125		996.485	728.8831	QC'd by Asinex Ltd.
Activator	100	1997.1382	90	Partial curve; high efficacy	-4	4.045	0.9983	2014.3209	17.1826	2.1	0 0 0 0 0 0 0 0	1721.6418		3.4525		3.378		3.9823		9.3476		32.2065		71.663		507.9293	1721.6418	QC'd by Asinex Ltd.
Activator	7.9433	108.3653	83	Complete curve; high efficacy	-5.1	1.21	0.9975	113.1273	4.762	1.1	0 0 0 0 0 0 0 0	108.9859		5.3988		3.1641		3.6828		13.9146		28.6189		79.4348		108.9185	108.9859	QC'd by Sigma Chemical Company
Activator	25.1189	97.5277	81	Complete curve; high efficacy	-4.6	1.9673	0.9962	100.5707	3.043	1.1	0 0 0 0 0 0 0 0	95.2374		6.1891		3.7946		0.6673		2.3461		4.2715		27.8734		94.9565	95.2374	QC'd by InterBioScreen
Activator	25.0638	66.3237	80	Complete curve; high efficacy	-4.601	1.9673	0.9714	68.9031	2.5794	1.1	0 0 0 0 0 0 0 0 0 0 0	62.2861	3.0694		-0.8446	0.6548	0.8398		3.196	1.329	8.8431		9.8094	19.5142	63.2257		62.2861	QC'd by Labotest
Activator	31.6228	320.5034	80	Complete curve; high efficacy	-4.5	3.1925	0.9981	307.6914	-12.812	1.1	0 0 0 0 0 0 0 0	307.0772		-15.3847		-10.2656		-16.2349		-18.8242		1.5755		14.2479		288.2493	307.0772	QC'd by Asinex Ltd.
Activator	100	1250.3151	72	Partial curve; high efficacy	-4	3.2975	0.9891	1276.0835	25.7685	2.1	0 0 0 0 0 0 0 0	1037.4663		6.7624		6.9493		6.7276		9.5847		24.545		121.6531		390.6169	1037.4663	QC'd by Enamine
Activator	112.2018	1092.6255	71	Partial curve; high efficacy	-3.95	4.9549	0.9966	1125.0573	32.4318	2.1	0 0 0 0 0 0 0 0	942.2591		60.5087		46.3912		41.2663		36.1932		23.2191		15.6355		148.326	942.2591	QC'd by Chem Div
Activator	112.2018	1105.2997	70	Partial curve; high efficacy	-3.95	4.5045	0.9998	1105.0358	-0.2639	2.1	0 0 0 0 0 0 0 0	894.0419		1.4284		-3.389		-3.9616		-0.1864		0.7724		6.7469		165.5024	894.0419	QC'd by Chem Div
Activator	100	1065.593	68	Partial curve; high efficacy	-4	2.9523	0.9515	1111.0006	45.4076	2.1	0 0 0 0 0 0 0 0	881.7465		-2.6974		-2.1437		0.9503		6.9385		71.8385		203.0705		370.0237	881.7465	QC'd by ChemBridge
Activator	100	908.1597	63	Partial curve; high efficacy	-4	2.3031	0.984	930.2328	22.0731	2.1	0 0 0 0 0 0 0 0	707.9397		-2.3217		-0.4363		-1.256		2.3502		33.6888		101.7463		350.2479	707.9397	QC'd by InterBioScreen
Activator	112.2018	806.0599	62	Partial curve; high efficacy	-3.95	4.9549	0.9966	816.2016	10.1417	2.1	0 0 0 0 0 0 0 0	676.784		1.2715		3.3965		0.6738		5.3915		7.0921		42.5928		122.1389	676.784	QC'd by NCI Chemotherapeutic Agents Repository
Activator	112.2018	783.7924	62	Partial curve; high efficacy	-3.95	4.9549	0.9997	789.9311	6.1387	2.1	0 0 0 0 0 0 0 0	655.0009		7.2057		7.6589		2.2588		-0.3456		4.7899		11.3778		112.2218	655.0009	QC'd by Maybridge
Activator	100	816.1621	61	Partial curve; high efficacy	-4	3.2475	0.987	832.4554	16.2933	2.1	0 0 0 0 0 0 0 0	673.5076		-6.2485		1.8467		0.2586		3.8523		14.6827		81.096		254.4096	673.5076	QC'd by TimTec
Activator	100	704.5917	58	Partial curve; high efficacy	-4	3.2475	0.9989	709.3423	4.7506	2.1	0 0 0 0 0 0 0 0	571.1291		0.7768		-0.8073		-0.5195		2.8471		7.8694		21.5244		216.3485	571.1291	QC'd by Chem Div
Activator	100	588.6904	55	Partial curve; high efficacy	-4	4.9549	0.9978	589.4841	0.7937	2.1	0 0 0 0 0 0 0 0	528.2116		-8.0271		-8.2801		-5.8459		-1.2817		0.2612		18.8362		129.6249	528.2116	QC'd by Specs

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：Burnham Center for Chemical Genomics 靶标：N/A

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

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

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

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

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

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

BatchID	%Activity_Corrected at 10 uM	Value at 10 uM	FRatio	Mean High	STD Deviation High	Mean Low	STD Deviation Low
MLS-0047618.P030	-3.49	3.6821	1.04	4.17	0.55	9.39	0.94
MLS-0047644.P030	5.01	4.1164	0.92	4.17	0.55	9.39	0.94
MLS-0047572.P030	-2.83	3.6969	1.04	4.17	0.55	9.39	0.94
MLS-0051226.P030	0.81	3.9451	0.97	4.17	0.55	9.39	0.94
MLS-0018734.P030	-4.41	3.6865	1.04	4.17	0.55	9.39	0.94
MLS-0099666.P028	1.27	3.9809	0.98	4.17	0.55	9.39	0.94
MLS-0021904.P031	-3.01	3.7812	1.01	4.17	0.55	9.39	0.94
MLS-0003494.P030	6.80	4.2136	0.88	4.17	0.55	9.39	0.94
MLS-0041706.P030	4.32	4.1571	0.93	4.17	0.55	9.39	0.94
MLS-0051069.P030	-3.40	3.7722	0.98	4.17	0.55	9.39	0.94
MLS-0008767.P030	-4.32	3.6982	1.10	4.17	0.55	9.39	0.94
MLS-0004317.P030	-5.10	3.6501	1.05	4.17	0.55	9.39	0.94
MLS-0024446.P030	1.54	3.976	1.00	4.17	0.55	9.39	0.94
MLS-0043221.P030	3.45	4.1364	0.94	4.17	0.55	9.39	0.94
MLS-0093353.P028	2.20	4.0861	0.93	4.17	0.55	9.39	0.94
MLS-0039240.P030	-3.25	3.8052	0.98	4.17	0.55	9.39	0.94
MLS-0009783.P025	0.25	3.893	0.94	4.17	0.55	9.39	0.94
MLS-0027652.P031	1.20	3.9291	0.96	4.17	0.55	9.39	0.94
MLS-0034571.P030	-0.40	3.8585	0.96	4.17	0.55	9.39	0.94
MLS-0001714.P030	3.94	4.0825	0.92	4.17	0.55	9.39	0.94

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：Broad Institute 靶标：N/A

External ID: 2084-01_Activator_SinglePoint_HTS_Activity

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

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

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

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

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

EXPECTED OUTCOME: Active compounds result in increasing readout signal.
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 80.

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

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

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

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

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

REPRODUCIBILITY_COSINE_TRANSFORM	REPLICATE_A_ACTIVITY_SCORE_12.5uM_(%)	REPLICATE_B_ACTIVITY_SCORE_12.5uM_(%)
0.803	5.022	0.742
0.999	-18.176	-19.61
0.862	-7.599	-1.979
0.757	-0.821	-11.086
0.99	28.589	38.087
0.996	-6.025	-4.98
0.937	16.619	7.568
0.739	7.024	0.325
0.999	-2.618	-2.419
0.633	-4.724	0.472
1	-14.15	-15.053
0.9	2.327	0.809
0.842	2.56	0.563
0.351	-7.412	16.314
0.7	-10.131	0.108
0.996	-27.692	-23.156
0.698	-11.06	0.138
0.989	11.846	8.737
0.715	0.101	8.982
1	-95.322	-95.121

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：DNA polymerase iota [Homo sapiens]

External ID: PolI100

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

Comment: Compound Ranking:

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

Phenotype	Potency	Efficacy	Curve_Description	Fit_LogAC50	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.447 uM	Activity at 2.333 uM	Activity at 11.29 uM	Activity at 58.90 uM	Activity at 114.8 uM	Compound QC
Activator	100	38.2868	Single point of activity	-4	4.9549	0.9173	40	1.7132	3	0 0 0 0 0	30.0132	5.2309	2.1349	2.7977	-1.489	30.0132	QC'd by "Asinex Ltd."
Activator	100	54.6995	Single point of activity	-4	4.9549	0.9067	56.2988	1.5993	3	0 0 0 0 0	42.8693	6.5044	4.3471	-0.522	-3.4058	42.8693	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	-17.2476	21.6169	12.7311	22.8702	20.8427	-17.2476	QC'd by "Asinex Ltd."
Activator	100	74.7794	Single point of activity	-4	4.9549	0.9041	76.0108	1.2314	3	0 0 0 0 0	57.8804	6.7166	3.0104	3.7559	-6.8905	57.8804	QC'd by "Asinex Ltd."
Activator	100	53.1278	Single point of activity	-4	4.9549	0.9205	64.5427	11.4149	3	0 0 0 0 0	51.5124	12.0687	10.6696	16.4441	7.3638	51.5124	QC'd by "Asinex Ltd."
Activator	89.1251	88.3489	Single point of activity	-4.05	4.9549	0.8504	83.4345	-4.9144	3	0 0 0 0 0	66.09	-2.4773	2.699	3.4126	-18.2031	66.09	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	28.3132	7.6044	7.1621	1.8797	-18.6884	28.3132	QC'd by "Asinex Ltd."
Inactive									4	0 0 0	-12.1963	15.5341	6.1257			-12.1963	QC'd by "Asinex Ltd."
Activator	70.7946	131.9739	Single point of activity	-4.15	4.9549	0.9814	124.2791	-7.6948	3	0 0 0 0	113.1868	-14.9551	3.8059	-12.5697		113.1868	QC'd by "Asinex Ltd."
Activator	50.1187	98.8583	Partial curve; high efficacy; poor fit	-4.3	2.8473	0.9993	127.1199	28.2616	2.3	0 0 0	118.3612	29.7145	27.0534			118.3612	QC'd by "Asinex Ltd."
Activator	89.1251	52.8332	Single point of activity	-4.05	4.9549	0.8839	46.5982	-6.2351	3	0 0 0 0 0	35.9582	-5.7785	0.0051	-3.002	-11.6645	35.9582	QC'd by "Asinex Ltd."
Activator	44.6684	58.1237	Single point of activity	-4.35	3.132	1	63.6237	5.5	3	0 0 0	60.693	5.2497	5.4646			60.693	QC'd by "Asinex Ltd."
Activator	100	32	Partial curve; partial efficacy; poor fit	-4	4.9549	0.7933	42	10	2.4	0 0 0 0 0	31.927	6.4109	17.9669	11.7318	10.1379	31.927	QC'd by "Asinex Ltd."
Activator	70.7946	52.3872	Partial curve; partial efficacy; poor fit	-4.15	3.132	0.9366	88.8106	36.4234	2.4	0 0 0	79.2341	43.0009	30.195			79.2341	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	10.1042	-2.0932	-3.6864	-11.1533	-2.2306	10.1042	QC'd by "Asinex Ltd."
Activator	35.4813	94.2201	Single point of activity	-4.45	1.7137	1	103.2201	9	3	1 0 0	91.9965	33.4991	9.8887			91.9965	QC'd by "Asinex Ltd."
Activator	89.1251	188.8272	Single point of activity	-4.05	4.9549	0.9819	184.3761	-4.4511	3	0 0 0 0 0	142.9272	-4.9415	0.8751	3.841	0.2429	142.9272	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	15.1304	-16.5373	-21.4322	-21.4132	-27.9881	15.1304	QC'd by "Asinex Ltd."
Activator	89.1251	54.5907	Single point of activity	-4.05	4.9549	0.8982	43.8763	-10.7143	3	0 0 0 0 0	34.3843	-5.7785	-4.8931	-14.2749	-14.7248	34.3843	QC'd by "Asinex Ltd."
Activator	56.2341	79.3018	Partial curve; high efficacy; poor fit	-4.25	3.132	0.9915	106.1029	26.8011	2.3	0 0 0	98.2435	30.6599	23.0391			98.2435	QC'd by "Asinex Ltd."

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：

External ID: epac2-activator-v2

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

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

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

Phenotype	Potency	Efficacy	Curve_Description	Fit_LogAC50	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.120 uM	Activity at 0.611 uM	Activity at 3.058 uM	Activity at 15.29 uM	Activity at 75.76 uM	Activity at 149.6 uM	Compound QC
Inactive					4.9549	0.8563	-9.9979	-3.182	4	0 0 0 0 0 0	-8.823	-2.9167	-5.5515	-1.8183	-11.2482	-10.2408	-8.823	QC'd by Microsource
Inactive					1.5386	0.564	4	-4.8546	4	0 0 0 0 0 1	-7.5471	-0.6835	-9.0455	-3.7039	-1.1903	3.153	-7.5471	QC'd by Microsource
Inactive					4.9549	0.431	-21.0949	2.1993	4	0 0 0 0 0 0	-29.6624	-1.0839	-27.7771	-29.2982	-9.5254	-9.846	-29.6624	QC'd by Microsource
Inactive					4.9549	0.3341	-1.3603	-6.9124	4	0 0 0 0 0 1	-15.5699	-9.927	-2.5767	-5.8552	-8.5221	-2.3836	-15.5699	QC'd by Microsource
Inhibitor	0.8913	14.1351	Complete curve; partial efficacy; poor fit	-6.05	2.1211	0.6534	-22.6728	-8.5377	-1.4	0 0 0 0 0 0	-21.1521	-8.7814	-12.9514	-21.2535	-30.9774	-17.0722	-21.1521	QC'd by Microsource
Inactive					3.132	0.4725	0.5	-4.2284	4	0 0 0 0 0 0	-0.2612	-1.9763	-2.0592	-8.107	-2.1288	0.8201	-0.2612	QC'd by Microsource
Inactive					4.9549	0.4917	-10.967	-2.3149	4	0 0 0 0 0 0	-10.1405	-1.5124	-2.5506	-5.4129	-17.8892	-4.0897	-10.1405	QC'd by Microsource
Inactive					3.99	0.5924	-19.9348	0	4	0 0 0 0 0 0	-28.6957	-6.2302	5.7918	-15.0449	-23.6413	-7.309	-28.6957	QC'd by Microsource
Inactive					4.9549	0.4102	-6.1308	5	4	0 0 0 0 0 0	-4.355	0.8289	8.6021	-2.8349	-16.359	2.161	-4.355	QC'd by Microsource
Inactive					4.9549	0.369	-1.5	-14.3369	4	0 0 0 0 0 1	-9.3021	-12.7808	2.0015	-0.2528	-12.5875	4.4401	-9.3021	QC'd by Microsource
Inhibitor	0.7079	23.6221	Complete curve; partial efficacy; poor fit	-6.15	3.0654	0.6513	-24.5288	-0.9067	-1.4	0 0 0 0 0 1	-13.9864	-1.5889	-9.6741	-27.4469	-33.3677	-12.1786	-13.9864	QC'd by Microsource
Inactive									4		-22.4775	-11.6889	-11.6628	-13.5576	-20.9035	-8.6191	-22.4775	QC'd by Microsource
Inactive					0.8	0.7088	-25.4933	-11.6918	4	0 0 0 0 0 0	-24.5778	-16.5266	-8.0765	-17.2318	-20.0221	-22.1348	-24.5778	QC'd by Microsource
Inhibitor	0.1778	19.9985	Complete curve; partial efficacy; poor fit	-6.75	4.9549	0.6578	-21.9347	-1.9361	-1.4	0 0 0 0 0 0	-23.6561	-4.5301	-30.7789	-20.7284	-19.2667	-15.4849	-23.6561	QC'd by Microsource
Inactive					1	0.9184	-24.9585	-2.7331	4	0 0 0 0 0 1	-15.8401	-9.7776	-20.4013	-20.4952	-26.2154	-25.1202	-15.8401	QC'd by Microsource
Inactive									4		-16.8662	-15.0798	-16.9114	-17.163	-13.1564	-16.5558	-16.8662	QC'd by Microsource
Inactive									4		7.7575	3.3126	6.1383	6.2047	-2.0209	6.2212	7.7575	QC'd by Microsource
Inactive									4		3.7597	9.7909	7.0224	3.0975	-6.8965	12.3146	3.7597	QC'd by Microsource
Inactive					4.9549	0.6335	-4.9129	6	4	0 0 0 0 0 0	-8.8467	6.1191	5.3496	5.0731	-9.0941	2.984	-8.8467	QC'd by Microsource
Inactive					4.9549	0.7097	-7.2734	0	4	0 0 0 0 0 0	-5.915	1.1538	-1.0587	0.2688	-11.4778	-4.1213	-5.915	QC'd by Microsource

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：N/A

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

Inhibition at 4.8 uM
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19
11.19

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：DNA polymerase eta [Homo sapiens]

External ID: PolE100

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

Comment: Compound Ranking:

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

Phenotype	Potency	Efficacy	Curve_Description	Fit_LogAC50	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.457 uM	Activity at 2.290 uM	Activity at 11.40 uM	Activity at 57.10 uM	Activity at 114.0 uM	Compound QC
Inactive									4		-18.6944	-16.4688	-21.0535	-18.6569	-21.2387	-18.6944	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-10.5731	-6.3238	-5.0728	-10.0177	-9.1591	-10.5731	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-20.9106	-9.1883	-14.5238	-10.32	-16.7599	-20.9106	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-14.976	-6.1336	-5.9392	-8.0291	-13.3224	-14.976	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-17.0295	-7.3413	-7.7338	-7.023	-12.9903	-17.0295	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-7.6456	7.5602	6.2602	5.948	3.5763	-7.6456	QC'd by "Chem Div"
Inhibitor	56.2341	51.8151	Partial curve; partial efficacy; poor fit	-4.25	2.3332	0.9681	-53.5412	-1.7261	-2.4	0 0 0 0 0	-52.4914	-0.4336	-0.9826	-4.7322	-25.7264	-52.4914	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-11.9031	-7.9949	-12.5613	-13.3404	-9.3154	-11.9031	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-18.4236	0.258	0.8315	-1.6401	-6.8466	-18.4236	QC'd by "Chem Div"
Inactive									4		-17.2118	-16.2591	-19.8884	-17.4024	-20.0078	-17.2118	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-16.8313	-10.0087	-8.8391	-10.5867	-9.3418	-16.8313	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-6.7293	-13.0106	-9.8363	-10.4044	-13.1352	-6.7293	QC'd by "Chem Div"
Inactive									4		-0.6109	-0.508	3.969	1.3962	3.5402	-0.6109	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-19.2263	-10.4317	-10.645	-12.9544	-8.0548	-19.2263	QC'd by "Chem Div"
Inactive									4	0 0 0 0 1	-19.5782	-17.1915	-17.4143	-17.6927	-30.3966	-19.5782	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-17.5302	-9.8783	-9.1532	-13.5844	-9.2694	-17.5302	QC'd by "Chem Div"
Inactive									4		-11.9062	-9.7368	-9.5071	-10.0381	-13.0691	-11.9062	QC'd by "Chem Div"
Inactive									4		-19.9153	-18.2374	-18.6714	-22.0089	-21.7411	-19.9153	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	14.5935	1.829	2.4851	2.7485	-0.7044	14.5935	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-10.4477	-8.2332	-4.1692	-6.4251	-8.3536	-10.4477	QC'd by "Chem Div"

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：

External ID: epac2-inhibitor-v2

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

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

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

Phenotype	Potency	Efficacy	Curve_Description	Fit_LogAC50	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.00850 uM	Activity at 0.018 uM	Activity at 0.090 uM	Activity at 0.151 uM	Activity at 0.235 uM	Activity at 0.457 uM	Activity at 0.814 uM	Activity at 1.171 uM	Activity at 2.284 uM	Activity at 4.113 uM	Activity at 5.853 uM	Activity at 11.42 uM	Activity at 20.49 uM	Activity at 29.59 uM	Activity at 56.64 uM	Activity at 111.7 uM	Activity at 238.8 uM	Compound QC
Inactive					3.6272	0.8626	-16.9749	3	4	0 0 0 0 0 0	-11.3522		4.4122				-0.1869			3.8551			-9.0486			-22.4791	-11.3522		QC'd by SigmaAldrich
Inactive					1.21	0.9115	1	26.5	4	0 0 0 0 0 0	3.0879		22.5222				31.9661			22.8496			17.2717			6.5589	3.0879		QC'd by NCI
Inactive					0.3	0.7243	-12.8995	38	4	0 0 0 0 0 0	-10.7496	28.5168				21.9546			5.2096			6.0738			11.3009	-10.7496			QC'd by Prestwick Chemical; Inc.
Inactive					4.9549	0.8029	-15.6993	-1.5	4	0 0 0 0 0 0	-11.416		-1.5504				-1.249			-4.6581			-0.4266			0.4639	-11.416		QC'd by BIOMOL
Inactive					4.9549	0.6678	-24.4602	3.4359	4	0 0 0 0 0 1	-5.4651		2.872				2.434			-38.4104			-25.2406			-9.2436	-5.4651		QC'd by BIOMOL
Inactive					2.4064	0.4215	1	17	4	0 0 0 0 0 0	5.8729		19.3202				14.8828			8.6332			24.907			11.2979	5.8729		QC'd by BIOMOL
Inactive					0.6	0.7078	-8.3138	14.5	4	0 0 0 0 0 0	-10.7777		11.8187				1.9932			1.9062			-11.5115			-0.0866	-10.7777		QC'd by BIOMOL
Inactive					3.99	0.916	12.5	29	4	0 0 0 0 0 0	11.3216		25.3511				10.2782			12.6928			12.0425			15.0596	11.3216		QC'd by BIOMOL
Inactive					4.9549	0.7598	-8.0307	2	4	0 0 0 0 0 0	-6.9632		-1.9133				5.8317			-9.609			-8.7246			-6.018	-6.9632		QC'd by SigmaAldrich
Inactive					0.7	0.6402	-18.8089	-2.3735	4	0 0 0 0 0 0	-14.8407			-3.9662			-6.7181			-0.3112			-9.968			-9.2615	-14.8407		QC'd by Microsource
Inactive					4.9549	0.9739	-11.7501	2	4	0 0 0 0 0 0	-11.6929		3.078				0.2437			2.2683			-12.7084			-10.568	-11.6929		QC'd by Microsource
Inactive					0.5	0.7605	-11.0605	6	4	0 0 0 0 0 0	-14.6337		3.6876				-3.5123			-6.8473			-7.5675			-5.2666	-14.6337		QC'd by BIOMOL
Inactive									4		-7.53	4.7778				-6.7829			-15.1322			-23.649			9.0847	-7.53			QC'd by Prestwick Chemical; Inc.
Inactive					4.9549	0.6409	-3.2949	14	4	0 0 0 0 0 1	12.9818		17.651				4.102			20.6219			2.497			-3.5791	12.9818		QC'd by BIOMOL
Inactive					1.8265	0.7407	-32.7287	-10.9373	4	0 0 0 0 0 0	-28.3802		-13.7631				-19.1044			-5.7811			-12.8137			-32.2739	-28.3802		QC'd by Tocris
Activator	39.8107	46.538	Single point of activity	-4.4	4.4495	0.7454	56.6458	10.1078	3	0 0 0 0 0 1	17.1415		13.5032				25.5832			14.464			6.9538			49.1102	17.1415		QC'd by SigmaAldrich
Inactive					4.9549	0.5359	-17.7206	3.5	4	0 0 0 0 0 0	-16.8505		-6.866				13.1002			-5.4009			12.3928			-11.0619	-16.8505		QC'd by SigmaAldrich
Inactive					4.9549	0.657	1.1082	10.5	4	0 0 0 0 0 1	10.7172		11.3518				5.1253			15.2488			-0.3265			2.2476	10.7172		QC'd by Prestwick Chemical; Inc.
Inactive					0.7	0.842	-14.4407	10.5	4	0 0 0 0 0 0	-8.7006				13.0238			6.2792			7.2892			3.5691			0.9878	-8.7006	QC'd by BIOMOL
Inactive					1.01	0.8718	2.4065	-26.9475	4	0 0 0 0 0 1	-20.1909		-25.9748				-30.373			-20.9376			-21.2882			-7.9946	-20.1909		QC'd by Prestwick Chemical; Inc.

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：Broad Institute 靶标：hedgehog, isoform A [Drosophila melanogaster]

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

REPRODUCIBILITY_COSINE_TRANSFORM	REPLICATE_A_ACTIVITY_SCORE_30.34uM_(%)	REPLICATE_B_ACTIVITY_SCORE_30.34uM_(%)
0.9766	13.043	8.333
0.9926	13.043	16.667
0.9594	9.091	16.667
0.9991	18.182	16.667
0.7071	0	9.091
0.7071	8.333	0
0.9594	16.667	9.091
0.9594	-16.667	-9.091
0.9991	8.333	9.091
0.9991	8.333	9.091
0.9991	8.333	9.091
0.7071	8.333	0
0.9878	25	18.182
1	8.333	8.333
0.9487	8.333	16.667
1	16.667	16.667
1	16.667	16.667
0.9487	8.333	16.667
0.9914	21.739	16.667
0.9991	18.182	16.667

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：23209 靶标：N/A

External ID: UIHTS20180925

Protocol: Assay overview:
The purpose of this assay is to identify test compounds that differentially kill either epithelial cells or mesenchymal cells, critical components of EMT. The PC-3E+ and TEM4-18 cell lines, characterized as epithelial cells and mesenchymal cells respectively, were established in Henry Lab (Ref 1). For potential discriminative modulators of EMT, the two cells lines were labeled with GFP and mCherry respectively, and co-cultured together to eliminate those hits that non-discriminatingly kill both epithelial cells and mesenchymal cells by cytotoxicity irrelevant to the EMT properties.
Protocol for the EMT project:
The primary screening data were generated as described in HTS assay protocol table (Ref 2).

Table 1: HTS assay protocol table
Step Parameter Value Description
1. Plate cells 200 uL By MultiFlo. PC3E GFP 2,000 cells/ well and TEM418 mCherry 2,000 cells/well (1:1). Overnight incubation
2. Remove media Flip the plate
3. Add fresh media 150 uL By MultiFlo
4. Controls 200 uL C01-H01, C12 -H12, Hygromycin dose response from H to C were positive control. Wells A01, B01, A12 and B12 were negative control
5. Library 50 uL MSSP library 1 uM final from middle plate in full media
6. Incubation 72 hrs 37 C and 5% CO2
7. Assay readout GFP and mCherry channel imaging Perkin Elmer Operetta high content imaging system, with Harmony image analysis software.
8. Image analysis Green cells and Read cells counting Normalized to the average cell number of well B1 and B12 (no inhibition of cell viability) controls on each plate to get relative cell viability for each well.
9. Hit selection 11 hits The hit criteria for preferential cell killing are compounds that Redcells_Normalized is lower or equal 0.41, GreenCells_Normalized is higher or equal
0.55, RedCells/GreenCells_Normalized ratio is lower or equal 0.65, and Non-fluorescent by itself for Red cell killing OR Greencells_Normalized is
lower or equal 0.41, RedCells_Normalized is higher or equal 0.46, RedCells/GreenCells_Normalized ratio is higher or equal 1.8 and Non-fluorescent
by itself for Green cell killing.

The primary screening data (imaging data) were translated into Green or Red cell numbers in each well in the data file.

Comment: Preferential killing compounds are considered as active hits (score of 100) when meeting one of two groups of criteria:

1. Compounds that Redcells_Normalized is lower or equal 0.41, GreenCells_Normalized is higher or equal 0.55, RedCells/GreenCells_Normalized ratio is lower or equal 0.65, and Non-fluorescent by itself for Red cell killing.

OR

2. Greencells_Normalized is lower or equal 0.41, RedCells_Normalized is higher or equal 0.46, RedCells/GreenCells_Normalized ratio is higher or equal 1.8 and Non-fluorescent by itself for Green cell killing.

No. of GreenCells	No. of RedCells	GreenCells_Normalized	RedCells_Normalized	RedCells/GreenCells_Normalized
1789	1180	0.88	1.08	1.22
1961	1081	0.97	0.99	1.02
1876	1291	0.93	1.18	1.27
2244	1094	1.11	1	0.9
2275	1210	1.12	1.11	0.98
1995	1398	0.98	1.28	1.3
2123	1293	1.05	1.18	1.13
2281	1227	1.13	1.12	1
1964	1090	0.97	1	1.03
2110	1295	1.04	1.18	1.14
2342	1361	1.16	1.24	1.08
2014	1316	0.99	1.2	1.21
2021	1270	1	1.16	1.16
2160	1287	1.07	1.18	1.1
2022	1197	1	1.09	1.1
1798	1082	0.89	0.99	1.11
1938	1199	1.08	1.06	0.98
2000	1260	1.12	1.11	0.99
1971	1201	1.1	1.06	0.96
2100	1130	1.17	1	0.85

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：DNA polymerase kappa [Homo sapiens]

External ID: PolK100

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

Comment: Compound Ranking:

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

Phenotype	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.477 uM	Activity at 2.393 uM	Activity at 11.99 uM	Activity at 60.11 uM	Activity at 107.2 uM	Compound QC
Inactive	4	0 0 0 0 0	1.4694	-3.5669	-6.235	2.8586	1.8042	1.4694	QC'd by "Chem Div"
Inactive	4	0 0 0 0 0	-4.2631	8.2218	8.0811	10.2927	-3.9947	-4.2631	QC'd by "Chem Div"
Inactive	4	0 0 0 0 0	6.0369	0.3398	-2.1048	-8.1695	-3.6822	6.0369	QC'd by "Chem Div"
Inactive	4		-2.0565	1.7294	-3.5894	-1.2575	-0.5402	-2.0565	QC'd by "Chem Div"
Inactive	4	0 0 0 0 1	2.3149	1.0048	4.6369	-1.9963	-3.3543	2.3149	QC'd by "Chem Div"
Inactive	4		7.2748	7.1515	6.1372	1.5197	5.2332	7.2748	QC'd by "Chem Div"
Inactive	4	0 0 0 0 0	1.006	-3.3873	-7.786	-9.3037	-9.1761	1.006	QC'd by "Chem Div"
Inactive	4	0 0 0 0 0	-0.0368	-9.4458	-10.5155	-9.0065	-12.9141	-0.0368	QC'd by "Chem Div"
Inactive	4	0 0 0 0 0	2.6	-7.8084	-12.3007	-2.0954	-6.6887	2.6	QC'd by "Chem Div"
Inactive	4	0 0 0 0 0	-11.4867	-18.9051	-17.4955	-19.0735	-9.6682	-11.4867	QC'd by "Chem Div"
Inactive	4	0 0 0 0 0	-7.5605	-17.2173	-11.0038	-16.5656	-22.4025	-7.5605	QC'd by "Chem Div"
Inactive	4		-7.5451	-1.1939	-1.3084	-5.8268	-5.3206	-7.5451	QC'd by "Chem Div"
Inactive	4	0 0 0 0 1	-5.5852	-4.3753	-1.0046	-3.1641	-10.1524	-5.5852	QC'd by "Chem Div"
Inactive	4	0 0 0 0 0	1.1172	-6.0391	7.0118	9.0446	1.6533	1.1172	QC'd by "Chem Div"
Inactive	4		2.3359	1.2518	1.6626	-0.9325	-0.9194	2.3359	QC'd by "Chem Div"
Inactive	4	0 0 0 0	-19.5354	0.3984	-4.1147	2.1883	-19.5354		QC'd by "Chem Div"
Inactive	4		-5.6552	-4.6769	-1.9378	-0.5867	-3.224	-5.6552	QC'd by "Chem Div"
Inactive	4		-11.3738	-10.4148	-13.8912	-10.4252	-7.8961	-11.3738	QC'd by "Chem Div"
Inactive	4		-6.1571	-8.7102	-2.9113	-5.2229	-3.4369	-6.1571	QC'd by "Chem Div"
Inactive	4	0 0 0 0 1	-7.3803	-8.8177	-11.1654	-6.5301	-15.9483	-7.3803	QC'd by "Chem Div"

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：N/A

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.

Phenotype	Potency	Efficacy	Curve_Description	Fit_LogAC50	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.074 uM	Activity at 0.369 uM	Activity at 1.840 uM	Activity at 9.233 uM	Activity at 46.10 uM	Compound QC
Inactive									4	0 0 0 0 0	0.2596	10.769	4.1255	-1.6909	-0.7487	0.2596	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-0.8876	-5.2018	-3.6707	0.3303	2.9155	-0.8876	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-4.2306	-10.0984	-0.7957	-0.9322	2.0609	-4.2306	QC'd by "Chem Div"
Inactive									4		5.8218	-1.6618	-3.0553	9.7773	-4.173	5.8218	QC'd by "Chem Div"
Inactive									4		-3.2651	11.605	-17.8848	5.9785	14.3087	-3.2651	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-7.241	3.2008	3.9728	-4.5121	3.9811	-7.241	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-9.807	8.9869	0.3484	0.3728	7.0197	-9.807	QC'd by "Chem Div"
Cytotoxic	17.7828	35.5846	Partial curve; partial efficacy	-4.75	2.3031	0.9974	-42.6167	-7.0321	-2.2	0 0 0 0 0	-39.1036	-6.2767	-6.4175	-8.2439	-13.6777	-39.1036	QC'd by "Chem Div"
Cytotoxic	3.5481	40.0619	Single point of activity	-5.45	4.9549	0.8999	-40.3659	-0.3039	-3	0 0 0 0 1	2.6367	-8.333	7.8061	-1.7484	-40.2332	2.6367	QC'd by "Chem Div"
Inactive									4	0 0 0 0 1	0.5424	1.6591	9.6647	14.2749	15.5896	0.5424	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	5.9628	-8.298	-2.3104	6.1361	-3.4428	5.9628	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-1.0151	-4.6247	-5.8885	-4.492	-0.7127	-1.0151	QC'd by "Chem Div"
Inactive									4		-0.9022	-1.2889	13.9053	-1.079	4.3101	-0.9022	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-23.5202	-1.5751	7.1469	-12.6721	9.6037	-23.5202	QC'd by "Chem Div"
Inactive									4	0 0 0 0 1	-0.075	-0.6173	-0.8732	5.135	2.1913	-0.075	QC'd by "Chem Div"
Cytotoxic	35.4813	33.3813	Single point of activity	-4.45	4.9549	0.4913	-37.3813	-4	-3	0 0 0 0 0	-30.3178	-0.6381	-23.6633	-3.8386	6.0591	-30.3178	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-17.414	0.1464	-4.8771	-5.0687	-7.6162	-17.414	QC'd by "Chem Div"
Inactive									4		-4.6673	-7.1501	-3.3264	-4.1232	-3.249	-4.6673	QC'd by "Chem Div"
Inactive									4	0 0 0 0 0	-17.3878	6.5726	2.9374	-7.8375	-3.1433	-17.3878	QC'd by "Chem Div"
Inactive									4	0 0 0 0 1	-10.2269	-7.0609	-5.5812	-5.8217	2.0518	-10.2269	QC'd by "Chem Div"

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：tyrosyl-DNA phosphodiesterase 2 [Homo sapiens]

External ID: TPD2_INH_EPIABS_1536_1X%INH PRUN

Protocol: Assay Overview:

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

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

Protocol Summary:

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

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

EPIABS = -log10( sample / background)

Where:

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

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

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

Where:

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

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

PubChem Activity Outcome and Score:

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

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

List of Reagents:

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

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

Inhibition at 12.8 uM
9.44
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43
9.43

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：Phospholipase C, gamma 1 [Homo sapiens]

External ID: PLCG1_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of the activity of phospholipase C isozymes, PLC-G1. In this assay, PLC-G1 isozyme is incubated with test compounds and fluorogenic reporter WH-15. As designed, test compounds that act as PLC-G1 inhibitors will prevent the hydrolysis of WH-15 fluorogenic reporter, thus preventing the release of IP3, a quinomethide derivative, and 6-aminoquinoline, which is highly fluorescent, leading to decreasing well fluorescence. Compounds are tested in singlicate at a nominal test concentration of 12.2 micromolar.

Protocol Summary:

Prior to the start of the assay, 2 microliters of PLC-G1 at a final concentration of 5pg/ul (in 50 mM HEPES pH 7.2, 70 mM KCl, 3mM CaCL2, 3mM EGTA, 2mM DTT, 0.04mg/mL acid-free BSA, with Cholate 0.5%) are dispensed into 1536 microtiter plates, 1 microliter of assay buffer is dispensed into columns 4-48 and 1 microliter of 0.2M EGTA is added to columns 1-3. Compounds are added to plate (final concentration 12.2uM) and incubated for 10 minutes at 25 degrees Celsius. The assay start by the addition of 2 microliter of WH-15 fluorogenic reporter at a final concentration 10uM in Assay Buffer to all wells. Plates were centrifuged and after 90 min of incubation at 25 degrees Celsius fluorescence is measured at 355nm excitation and 535nm emmision.

The percent inhibition for each compound was calculated as follows:

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

Where:

Test_Compound is defined as wells containing PLCG1 in the presence of test compound and WH15 fluoreogenic reporter.
High_Control is defined as wells containing PLCG1, WH15 fluoreogenic reporter and EGTA.
Low_Control is defined as the median of the wells containing test compounds.

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

PubChem Activity Outcome and Score:

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

The activity score range for active compounds is 100-11, for inactive 11-0.

List of Reagents:

PLCG1 isozyme (Supplied by Assay Provider)
WH-15 fluorogenic reporter (Supplied by KXTBio)
HEPES (Fisher, BP310)
Sodium cholate hydrate (Sigma, C6445)
CaCl2 (Sigma, 06991)
EGTA (Fisher, O2783)
DTT (Fisher, BP172)
KCl (Sigma, P9541)
1536-well plates (Corning, part 7261)

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

Inhibition at 12.2 uM
4.43
4.43
4.43
4.43
4.43
4.43
4.43
4.43
4.43
4.43
4.43
4.43
4.43
4.43
4.43
4.42
4.42
4.42
4.42
4.42

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：Co-chaperonin GroES

External ID: CHEMBL4392938

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

Target ChEMBL ID: CHEMBL4106139
ChEMBL Target Name: GroEL/GroES
ChEMBL Target Type: PROTEIN COMPLEX - Target is a defined protein complex, consisting of multiple subunits
Relationship Type: D - Direct protein target assigned
Confidence: Direct protein complex subunits assigned

Standard Type	Standard Relation	Standard Value	Standard Units
Inhibition	=	100	%
Inhibition	=	72	%
Inhibition	=	66	%
Inhibition	=	98	%
Inhibition	=	99	%
Inhibition	=	96	%
Inhibition	=	98	%
Inhibition	=	81	%
Inhibition	=	98	%
Inhibition	=	100	%
Inhibition	=	99	%
Inhibition	=	99	%
Inhibition	=	89	%
Inhibition	=	102	%
Inhibition	=	94	%
Inhibition	=	79	%
Inhibition	=	49	%
Inhibition	=	96	%
Inhibition	=	99	%
Inhibition	=	97	%

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：ChEMBL 靶标：Co-chaperonin GroES

External ID: CHEMBL4392939

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2019
Volume: 29
Issue: 9
First Page: 1106
Last Page: 1112
DOI: 10.1016/j.bmcl.2019.02.028

Target ChEMBL ID: CHEMBL4106139
ChEMBL Target Name: GroEL/GroES
ChEMBL Target Type: PROTEIN COMPLEX - Target is a defined protein complex, consisting of multiple subunits
Relationship Type: D - Direct protein target assigned
Confidence: Direct protein complex subunits assigned

Standard Type	Standard Relation	Standard Value	Standard Units
Inhibition	=	98	%
Inhibition	=	86	%
Inhibition	=	85	%
Inhibition	=	66	%
Inhibition	=	89	%
Inhibition	=	101	%
Inhibition	=	101	%
Inhibition	=	94	%
Inhibition	=	100	%
Inhibition	=	99	%
Inhibition	=	101	%
Inhibition	=	77	%
Inhibition	=	62	%
Inhibition	=	100	%
Inhibition	=	93	%
Inhibition	=	65	%
Inhibition	=	87	%
Inhibition	=	80	%
Inhibition	=	101	%
Inhibition	=	98	%

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：Burnham Center for Chemical Genomics 靶标：cystic fibrosis transmembrane conductance regulator [Homo sapiens]

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

%Activity at 5 uM	Value	Mean Low	Std Deviation Low	Mean High	Std Deviation High
17.7	1388986	164067.88	8625.2	2080144.63	200104.91
-5.5	2023753	164067.88	8625.2	2080144.63	200104.91
-1.9	1947331	164067.88	8625.2	2080144.63	200104.91
4.9	1776516	164067.88	8625.2	2080144.63	200104.91
4.4	1681435	164067.88	8625.2	2080144.63	200104.91
7.1	1654104	164067.88	8625.2	2080144.63	200104.91
-16.9	2220014	164067.88	8625.2	2080144.63	200104.91
5.5	1825441	164067.88	8625.2	2080144.63	200104.91
-10.2	2135725	164067.88	8625.2	2080144.63	200104.91
13.1	1722580	164067.88	8625.2	2080144.63	200104.91
0.1	1996001	164067.88	8625.2	2080144.63	200104.91
-1.5	1994666	164067.88	8625.2	2080144.63	200104.91
12.6	1687553	164067.88	8625.2	2080144.63	200104.91
-12.4	2138337	164067.88	8625.2	2080144.63	200104.91
1.9	1864558	164067.88	8625.2	2080144.63	200104.91
14.3	1574274	164067.88	8625.2	2080144.63	200104.91
20.4	1414289	164067.88	8625.2	2080144.63	200104.91
67.4	531762	164067.88	8625.2	2080144.63	200104.91
3.1	1778225	164067.88	8625.2	2080144.63	200104.91
53.5	823431	164067.88	8625.2	2080144.63	200104.91

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：muscarinic acetylcholine receptor M1 [Homo sapiens]

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.

Inhibition at 3 uM
6.48
6.48
6.48
6.48
6.48
6.48
6.48
6.48
6.48
6.48
6.48
6.48
6.47
6.47
6.47
6.47
6.47
6.47
6.47
6.47

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：N/A

External ID: FBW7_ACT_ALPHA_1536_1X%ACT PRUN

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

The percent activation for each compound was calculated as follows:

100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Where:
Test_Compound is defined as wells containing RLFbw7 (mutant), cyclin E peptide and Nickel acceptor beads in the presence of test compound
High_Control is defined as wells containing WTFbw7 (wild type), cyclin E peptide and Nickel acceptor beads
Low_Control is defined as the median of the wells containing DMSO, RLFbw7 (mutant), cyclin E peptide and Nickel acceptor beads
PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine active compounds. Two values were calculated: (1) the average percent activation of all compounds tested for the screen, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater percent activation than the cutoff parameter (1.85% in the case here) was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The activity score range for active compounds is 100-1, for inactive 1-0.
List of Reagents:
Ni Beads- PerkinEmer Lifesciences Cat#6760619R
RLFbw7-Assay Provider
WTFbw7-Assay Provider
Cyclin E peptide-Assay Provider
5M NaCl- Sigma Cat# S6546-1L
Tween20- Fisher Cat# BP337
Tris 1M pH7.4 Research Organics Cat# 9686T
BSA-Sigma Cat#A7030
?-Mercaptoethanol-SigmaM6250
1536-well plates (Corning, part 7254)

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

Inhibition at 26.1 uM
1.22
1.22
1.22
1.22
1.21
1.21
1.21
1.21
1.21
1.21
1.21
1.21
1.21
1.21
1.21
1.21
1.21
1.21
1.21
1.21

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：Smad3 [Homo sapiens]

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.

Phenotype	Potency	Efficacy	Curve_Description	Fit_LogAC50	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 0.074 uM	Activity at 0.369 uM	Activity at 1.840 uM	Activity at 9.231 uM	Activity at 46.10 uM	Compound QC
Inactive									4	0 0 0 0 0	27.0569	9.9398	10.1515	0.1671	5.5721	27.0569	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 1	-4.9362	-9.414	12.0824	-11.0493	-7.696	-4.9362	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 1	5.9595	4.342	-1.5624	-2.6449	-8.9538	5.9595	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	-19.7473	-1.448	7.5701	-38.1554	-17.3097	-19.7473	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	-1.2351	-5.5487	-5.0573	-16.6211	2.7653	-1.2351	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	7.1959	-7.7682	4.4899	3.3992	13.3707	7.1959	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 1	8.9833	15.335	4.2535	4.1946	-14.3236	8.9833	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	7.9022	-10.5174	13.4936	-10.4686	7.2323	7.9022	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	-11.8347	12.2839	-2.7256	-19.2666	-5.8034	-11.8347	QC'd by "Asinex Ltd."
Inhibitor	35.4813	106.2444	Single point of activity	-4.45	4.4495	0.9934	-109.7251	-3.4808	-3	0 0 0 0 0	-84.6645	-7.4849	-2.0755	-4.8114	0.1432	-84.6645	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	-3.6	-2.0717	4.9414	15.4055	-0.2463	-3.6	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	0.7641	0	28.3456	12.1698	0.9078	0.7641	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 1	-3.7338	-9.9559	0.3986	8.9255	12.5033	-3.7338	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	-1.7797	3.883	1.182	-4.185	1.7497	-1.7797	QC'd by "Asinex Ltd."
Inhibitor	15.8489	38.9608	Single point of activity	-4.8	3.6772	0.9889	-35.4608	3.5	-3	0 0 0 0 0	-32.884	2.0677	5.819	2.7318	-1.3119	-32.884	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	-3.797	8.4821	-2.1836	12.76	5.4907	-3.797	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	-18.7499	1.0272	3.815	20.5199	1.7606	-18.7499	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	0.464	0	9.4101	-6.5206	0.9067	0.464	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 0	0.2371	9.7122	-4.6112	-6.6419	-3.2889	0.2371	QC'd by "Asinex Ltd."
Inactive									4	0 0 0 0 1	3.6799	4.8924	1.7621	-1.6686	-4.4945	3.6799	QC'd by "Asinex Ltd."

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：Burnham Center for Chemical Genomics 靶标：tyrosine-protein phosphatase non-receptor type 5 isoform a [Homo sapiens]

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

Protocol: STEP Assay HTS Protocol

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

%Activity at 20 uM_Norm	%Activity at 20 uM_Corr	Value	Mean Low	Std Deviation Low	Mean High	Std Deviation High
-5.0402	0.3	4284.91	1163.51	25.63	4134.15	121.83
-1.3316	2.6	4174.77	1163.51	25.63	4134.15	121.83
-4.8197	0.3	4278.36	1163.51	25.63	4134.15	121.83
1.7794	3.4	4082.38	1163.51	25.63	4134.15	121.83
-1.2535	-1.5	4172.45	1163.51	25.63	4134.15	121.83
-4.9399	-4.2	4281.93	1163.51	25.63	4134.15	121.83
-7.5121	-2.8	4358.32	1163.51	25.63	4134.15	121.83
-4.3378	0.1	4264.05	1163.51	25.63	4134.15	121.83
-6.0925	-2.1	4316.16	1163.51	25.63	4134.15	121.83
-6.1417	-3.1	4317.62	1163.51	25.63	4134.15	121.83
-6.5477	-3.9	4329.68	1163.51	25.63	4134.15	121.83
-3.7859	-1.3	4247.66	1163.51	25.63	4134.15	121.83
11.693	4.9	3787.96	1163.51	25.63	4134.15	121.83
-4.1853	-3.6	4259.52	1163.51	25.63	4134.15	121.83
1.9357	0.5	4077.74	1163.51	25.63	4134.15	121.83
-3.2081	-0.1	4230.5	1163.51	25.63	4134.15	121.83
-3.1425	1.1	4228.55	1163.51	25.63	4134.15	121.83
-5.8992	1.6	4310.42	1163.51	25.63	4134.15	121.83
-11.516	-0.4	4477.22	1163.51	25.63	4134.15	121.83
-12.486	-5.9	4506.02	1163.51	25.63	4134.15	121.83

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：nuclear receptor coactivator 3 isoform a [Homo sapiens]

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.

Inhibition at 3.6 uM
20.65
20.65
20.65
20.65
20.65
20.65
20.64
20.64
20.64
20.64
20.64
20.64
20.63
20.63
20.63
20.63
20.63
20.63
20.63
20.63

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：NCGC 靶标：RecName: Full=Rap guanine nucleotide exchange factor 3; AltName: Full=Exchange factor directly activated by cAMP 1; AltName: Full=Exchange protein directly activated by cAMP 1; Short=EPAC 1; AltName: Full=Rap1 guanine-nucleotide-exchange factor directly activated by cAMP; AltName: Full=cAMP-regulated guanine nucleotide exchange factor I; Short=cAMP-GEFI

External ID: EPAC1-agonist-p1

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

Comment: Compound Ranking:

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

Phenotype	Fit_HillSlope	Fit_R2	Fit_InfiniteActivity	Fit_ZeroActivity	Fit_CurveClass	Excluded_Points	Max_Response	Activity at 3.040 uM	Activity at 15.63 uM	Activity at 76.90 uM	Activity at 154.0 uM	Compound QC
Inactive	4.9549	0.8886	-12.2065	1.5	4	0 0 0 0	-9.3388	0.1709	0.8134	3.1053	-9.3388	QC'd by "Key Organics Ltd."
Inactive	4.4495	0.9946	1	9	4	0 0 0 0	1.3018	8.9908	8.9578	0.7198	1.3018	QC'd by "DPISMR"
Inactive	4.9549	0.6612	19	2	4	0 0 0 0	16.3973	7.6372	3.8451	0	16.3973	QC'd by "Key Organics Ltd."
Inactive	0.6	0.9546	18	-2	4	0 0 0 0	13.9071	1.7804	6.9386	9.7393	13.9071	QC'd by "DPISMR"
Inactive	4.9549	0.8159	-2.8337	4.5	4	0 0 0 0	-0.6947	6.0033	3.0043	3.9613	-0.6947	QC'd by "DPISMR"
Inactive	4.9549	0.8887	1	-15.0612	4	0 0 0 0	-2.3986	-15.051	-0.0139	4.4512	-2.3986	QC'd by "DPISMR"
Inactive	2.0937	0.9994	7	-0.9204	4	0 0 0 1	-0.3283	-0.767	0.3091	5.6555	-0.3283	QC'd by "DPISMR"
Inactive	4.9549	0.9917	11	3.5	4	0 0 0 1	2.8183	3.8289	3.2526	9.0005	2.8183	QC'd by "DPISMR"
Inactive	4.5045	0.987	15.5	8	4	0 0 0 0	14.5439	7.8628	8.5719	9.6022	14.5439	QC'd by "DPISMR"
Inactive	4.9549	0.4155	-0.5	-9.1434	4	0 0 0 1	-4.6031	-6.3695	3.3511	-4.639	-4.6031	QC'd by "DPISMR"
Inactive	4.5045	0.999	-0.7475	-8.2495	4	0 0 0 1	-5.4106	-6.4579	-0.6229	-0.8099	-5.4106	QC'd by "InterBioScreen"
Inactive					4		1.593	-1.4939	2.166	2.222	1.593	QC'd by "InterBioScreen"
Inactive	4.9549	0.6286	11	0.0586	4	0 0 0 0	8.6061	3.9411	-0.2733	-2.4512	8.6061	QC'd by "InterBioScreen"
Inactive	1.6259	0.9916	15	4	4	0 0 0 0	13.3001	3.6753	5.219	10.0023	13.3001	QC'd by "InterBioScreen"
Inactive	4.5045	0.9816	-6.5515	4	4	0 0 0 0	-5.0429	3.3448	4.7645	1.5692	-5.0429	QC'd by "DPISMR"
Inactive					4		6.4595	2.8715	3.5686	4.1617	6.4595	QC'd by "DPISMR"
Inactive	3.6772	0.9995	12.5	-2.1757	4	0 0 0 0	9.9965	-2.2298	-2.0336	2.0579	9.9965	QC'd by "DPISMR"
Inactive	4.5045	0.9996	-0.8312	7	4	0 0 0 1	3.5126	6.8746	6.6766	-0.6927	3.5126	QC'd by "DPISMR"
Inactive	1.8617	0.9999	-6.2131	2	4	0 0 0 1	-1.3966	1.7089	-1.1576	-5.5943	-1.3966	QC'd by "DPISMR"
Inactive					4		-5.9044	-3.6854	-6.4554	-5.89	-5.9044	QC'd by "DPISMR"

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：nuclear receptor coactivator 1 isoform 1 [Homo sapiens]

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.

Inhibition at 3.6 uM
18.31
18.31
18.31
18.31
18.31
18.31
18.31
18.3
18.3
18.3
18.29
18.28
18.28
18.28
18.28
18.27
18.27
18.27
18.27
18.27

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set16

来源：The Scripps Research Institute Molecular Screening Center 靶标：Phospholipase C, beta 3 (phosphatidylinositol-specific) [Homo sapiens]

External ID: PLCB3_INH_QFRET_1536_1X%INH PRUN

Protocol: Assay Overview:

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

Protocol Summary:

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

The percent inhibition for each compound was calculated as follows:

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

Where:

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

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

PubChem Activity Outcome and Score:

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

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

List of Reagents:

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

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

Inhibition at 12.2 uM
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58
2.58

475-25-2 靶点实验数据