External ID: CHRM1_AG_FLUO8_1536_1X%ACT PRUN

Protocol: Assay Overview:

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

Protocol Summary:

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

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

Ratio = I_Max / I_Min

Where:

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

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

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

Where:

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

PubChem Activity Outcome and Score:

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

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

The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 1-0.

List of Reagents:

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

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

External ID: CHEMBL4831941

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2021
Volume: 223
First Page: 113630
Last Page: 113630
DOI: 10.1016/j.ejmech.2021.113630

External ID: CHEMBL4831936

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: Eur J Med Chem
Year: 2021
Volume: 223
First Page: 113630
Last Page: 113630
DOI: 10.1016/j.ejmech.2021.113630

External ID: CHEMBL4831940

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: Eur J Med Chem
Year: 2021
Volume: 223
First Page: 113630
Last Page: 113630
DOI: 10.1016/j.ejmech.2021.113630

External ID: CHRM1_PAM_FLUO8_1536_1X%ACT PRUN

Protocol: Assay Overview:

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

Protocol Summary:

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

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

Ratio = I_Max / I_Min

Where:

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

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

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

Where:

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

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

PubChem Activity Outcome and Score:

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

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

List of Reagents:

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

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

External ID: CHEMBL2182848

Protocol: N/A

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

Journal: J. Med. Chem.
Year: 2012
Volume: 55
Issue: 23
First Page: 10523
Last Page: 10531
DOI: 10.1021/jm301124s

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: SMAD3201

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

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

Comment: Compound Ranking:

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

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

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

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

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

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

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

IV. Reading plates:

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

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

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

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

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

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

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

External ID: CHRM1_ANT_FLUO8_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that act as antagonists and decrease activity of the human M1 muscarinic receptor (CHRM1; M1) that have been pre-treated with a known agonist, with the end result being a decrease in intracellular calcium. In this assay, CHO-K1 cells stably expressing human M1 are loaded with the Fluo-8 calcium indicator dye. Compounds are added followed by treatment with the activator acetylcholine at a concentration that results in 80% activation (Ec80). As designed, compounds that act as CHRM1 antagonists will decrease calcium mobilization, resulting in decreased relative fluorescence of the indicator dye below that of the Ec80 of acetylcholine. Compounds are tested in singlicate at a final nominal concentration of 3 uM.

Protocol Summary:

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

The day before the assay 3000 cells in 3 uL of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 17-24 hours. Next, 2 uL of the fluorogenic Fluo-8 intracellular calcium indicator mixture (prepared according to the manufacturer's protocol) was added to each well. Plates were then incubated for 1 hour at 37 C, 5% CO2, and 95 % RH, followed by 30 minute incubation at room temperature. Then, 15 nL of test compound in DMSO were transferred to appropriate wells. The assay was started by performing a basal read of plate fluorescence (470 - 495 nm excitation and 515 - 575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices) prior to all wells being treated with an EC80 concentration of acetylcholine. Then a real time fluorescence measurement was immediately performed for the remaining 140 seconds of the assay.

Hits for this assay were determined according to the following mathematical expression:

Ratio = I_Max / I_Min

Where:

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

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

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

Where:

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

PubChem Activity Outcome and Score:

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

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

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

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

List of Reagents:

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

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

External ID: SMAD3101

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

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

Comment: Compound Ranking:

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

External ID: CHEMBL4046178

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2017
Volume: 27
Issue: 18
First Page: 4482
Last Page: 4487
DOI: 10.1016/j.bmcl.2017.08.002

Target ChEMBL ID: CHEMBL4483242
ChEMBL Target Name: HBZY-1
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: APP-Toga-CHIKV-nsp2-p

Protocol: PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007)
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Control / Compound; 20 nL; Echo 655 acoustic dispenser, Greiner 1536-well solid bottom black plate.
2; Enzyme; 4 uL; BioRAPTR FRD liquid dispenser (Beckman Coulter).
3; Incubation; 15 min; room temperature.
4; Reagent; 4 uL; 2.5 uM Peptide 2 substrate.
5; Incubation; 1 hr; room temperature.
6; Detection; Fluorescence; WiewLux microplate reader (PerkinElmer), 525 nm excitation, 598/25 nm emission.

NOTES (numbers refer to sequence numbers above).
1. Briefly, 20 nL DMSO, positive control ZnAc (20nM final concentration), and test compounds were transferred into a 1,536-well solid bottom black plate (789176-F, Greiner One) via Echo 655 acoustic dispenser (Beckman Coulter). For primary screens, compounds were tested at 7 concentrations, 1:3 dilution points ranging from 25 uM to 34 nM. Follow-up confirmatory screens were carried out at 11 concentrations, 1:3 dilution points from 25 uM to 0.42 nM.
2. Four uL nsP2pro enzyme mix (150 nM final concentration) in 10 mM Tris-HCl pH 8.0 with 0.01% Tween 20 assay buffer was dispensed into the plate using a BioRAPTR FRD liquid dispenser (Beckman Coulter).
3. The plate was incubated at room temperature (protected from light) for 15 min
4. Four microliter of peptide 2 substrate (2.5 uM final concentration) in assay buffer was added to the plate.
5. After 1 hour, plates were immediately read on a ViewLux high-throughput CCD imager (Exposure = 10 sec, Gain = High, Speed = Slow, Binning = 2X). The above assay was also incorporated in the NCATS HTS facility41, which allowed for robotic liquid and compound dispensing, microplate handling, and fluorescence reading..

REFERENCE:
Inglese J, Shamu CE and Guy RK, Reporting data from high throughput screening of small molecule libraries, Nature Chemical Biology, 2007, 3(8): 438-441. doi.org/10.1038/nchembio0807-438.

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 [1].

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.

Reference:
1. Inglese J, Auld DS, Jadhav A, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A. 2006;103(31):11473-11478.

External ID: CHEMBL1799477

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: Eur. J. Med. Chem.
Year: 2011
Volume: 46
Issue: 7
First Page: 2797
Last Page: 2806
DOI: 10.1016/j.ejmech.2011.03.068

Target ChEMBL ID: CHEMBL3081
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL1942489

Protocol: N/A

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

Journal: Bioorg. Med. Chem.
Year: 2012
Volume: 20
Issue: 2
First Page: 832
Last Page: 840
DOI: 10.1016/j.bmc.2011.11.067

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4046197

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2017
Volume: 27
Issue: 18
First Page: 4482
Last Page: 4487
DOI: 10.1016/j.bmcl.2017.08.002

Target ChEMBL ID: CHEMBL4483242
ChEMBL Target Name: HBZY-1
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL4046195

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2017
Volume: 27
Issue: 18
First Page: 4482
Last Page: 4487
DOI: 10.1016/j.bmcl.2017.08.002

Target ChEMBL ID: CHEMBL4483242
ChEMBL Target Name: HBZY-1
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL4046196

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2017
Volume: 27
Issue: 18
First Page: 4482
Last Page: 4487
DOI: 10.1016/j.bmcl.2017.08.002

Target ChEMBL ID: CHEMBL4483242
ChEMBL Target Name: HBZY-1
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL4046194

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2017
Volume: 27
Issue: 18
First Page: 4482
Last Page: 4487
DOI: 10.1016/j.bmcl.2017.08.002

Target ChEMBL ID: CHEMBL4483242
ChEMBL Target Name: HBZY-1
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2013956

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: Eur. J. Med. Chem.
Year: 2012
Volume: 51
First Page: 216
Last Page: 226
DOI: 10.1016/j.ejmech.2012.02.045

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL643666

Protocol: N/A

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

Journal: J. Med. Chem.
Year: 1994
Volume: 37
Issue: 6
First Page: 787
Last Page: 792
DOI: 10.1021/jm00032a011

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous single protein target assigned

External ID: CHEMBL643668

Protocol: N/A

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

Journal: J. Med. Chem.
Year: 1997
Volume: 40
Issue: 5
First Page: 684
Last Page: 694
DOI: 10.1021/jm960594+

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4326712

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: 2018
Volume: 28
Issue: 23-24
First Page: 3712
Last Page: 3720
DOI: 10.1016/j.bmcl.2018.10.024

Target ChEMBL ID: CHEMBL1900
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL2390670

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 2013
Volume: 56
Issue: 11
First Page: 4135
Last Page: 4155
DOI: 10.1021/jm3017317

Target ChEMBL ID: CHEMBL372
ChEMBL Target Name: Homo sapiens
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL2013957

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: Eur. J. Med. Chem.
Year: 2012
Volume: 51
First Page: 216
Last Page: 226
DOI: 10.1016/j.ejmech.2012.02.045

External ID: CHEMBL848588

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1997
Volume: 40
Issue: 5
First Page: 684
Last Page: 694
DOI: 10.1021/jm960594+

External ID: CHEMBL4513082

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL4303835
ChEMBL Target Name: SARS-CoV-2
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: SARS-CoV-2 Screening Data

External ID: CHEMBL4004186

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2017
Volume: 27
Issue: 4
First Page: 887
Last Page: 892
DOI: 10.1016/j.bmcl.2017.01.006

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: BHood-p-21Jun2013-01

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

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

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

Comment:

External ID: CHEMBL4388106

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL614818
ChEMBL Target Name: HEK293
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL4388107

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL614818
ChEMBL Target Name: HEK293
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL4388108

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL614818
ChEMBL Target Name: HEK293
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL4388109

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL614818
ChEMBL Target Name: HEK293
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL4388096

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388097

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388098

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388099

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: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388088

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388089

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: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388090

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388091

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388092

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4004187

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: 2017
Volume: 27
Issue: 4
First Page: 887
Last Page: 892
DOI: 10.1016/j.bmcl.2017.01.006

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388093

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL1020264

Protocol: N/A

Comment: Journal: J. Nat. Prod.
Year: 2005
Volume: 68
Issue: 4
First Page: 620
Last Page: 622
DOI: 10.1021/np040199j

Target ChEMBL ID: CHEMBL1900
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388094

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: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388095

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388080

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL1900
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388081

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: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL1900
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4388082

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 168
First Page: 154
Last Page: 175
DOI: 10.1016/j.ejmech.2019.02.036

Target ChEMBL ID: CHEMBL2622
ChEMBL Target Name: Aldose reductase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4028923

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: Toxicol Sci
Year: 2013
Volume: 136
Issue: 1
First Page: 216
Last Page: 241
DOI: 10.1093/toxsci/kft176

Target ChEMBL ID: CHEMBL5918
ChEMBL Target Name: Canalicular multispecific organic anion transporter 2
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned