External ID: CHEMBL4360722

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: 178
First Page: 571
Last Page: 588
DOI: 10.1016/j.ejmech.2019.05.037

Target ChEMBL ID: CHEMBL2111385
ChEMBL Target Name: Cannabinoid receptor
ChEMBL Target Type: PROTEIN FAMILY - Target is a group of closely related proteins
Relationship Type: D - Direct protein target assigned
Confidence: Multiple direct protein targets may be assigned

External ID: JHICC_RGS_Act_HTS

Protocol: Assay overview:

To screen for compounds that activate the RGS4 protein, a HEK293 cell line which stably expresses M3R and inducibly expresses RGS4 is employed. RGS4 function is monitored by calcium flux with a commercially available Fluo4-AM dye. Compounds that do not show increase in the Fluo4 fluorescence in induced RGS4 expressed cells are considered as activator/potentiator hits. M3 receptor and other endogenous receptor activators/potentiators will be excluded through later counter-screening against non-induced parental cells.

Protocol for RGS4 Primary Screen:
1. Cell culture: Cells (HEK293-FlpIn-TREx/M3R/RGS4) are routinely cultured in DMEM (high glucose, w/ glutamine), 10%FBS, 1%Pen/Strep, 15ug/ml Blasticidin, 400ug/ml G418, 200ug/ml Hygromycin.
2. Cell plating: Add 50 ul/well of 200,000 cells/ml re-suspended in DMEM/high glucose medium with 10% FBS, 1% Pen/Strep. Include 10 ng/ml Doxycyclin (DOX) to induce RGS4 expression.
3. Incubate overnight at 37C and 5% CO2.
4. Remove medium and add 20 ul /well of 2uM Fluo4-AM solution to cells.
5. Incubate 30 minutes at 37C in incubator.
6. Prepare 6x compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer (HBSS-HEPES pH 7.4).
7. Remove Fluo4-AM dye solution and add 20 ul/well of assay buffer to cells.
8. Incubate 30 minutes at room temperature (RT).
9. Add 6x compounds in cell plates and incubate 20 minutes at RT.
9. Load cell plates on Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 10 seconds at 1Hz to establish baseline
11. After 100 seconds, add 4 ul of ECmax (carbachol) into the cell plates and record fluorescence for 100 seconds.
12. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout.
13. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors [26]
14. Calculate B scores [27] for test compounds using integrated ratios calculated in Step 12
15. Outcome assignment: If the B score of the test compound is less than 4 times the standard deviation (SD) of the B scores of integrated ratios of all library compounds above the mean (B score Activator Ratio16. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Int(((Log(ABS(B score ratio))-0.82)/0.44)*100), and they are normalized to the smallest and largest LOG(B score ratio), B score ratio, as in the result definition. The inactive test compounds are assigned a score of 0.


List of reagents

1. HEK293-FlpIn-TREx/M3R/RGS4 cell lines (provided by assay provider)
2. PBS: pH7.4 (Invitrogen Cat#10010049)
3. Medium: DMEM (Sigma, Cat#D5796)
4. Fetal Bovine Serum (Gemini, Cat# 100-106)
5. Hygromycin (Mediatech, Cat#30-240-CR)
6. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
7. Cell/stripper (Mediatech, Cat#25-056-Cl)
8. G418: (Invitrogen, Cat#11811-031)
9. Blasticidin (Sigma, Cat#R21001)
10. Doxycycline hyclate (Sigma, Cat#D9891)
11. HEPES (Sigma, Cat#H4034)
12. Fluo-4 (Invitrogen, Cat #F14202)
13. Pluronic F-127*20% in DMSO (Invitrogen, Cat#P-3000MP)
14. Atropine (Sigma, Cat#A0132)
15. Carbachol (Sigma, Cat# C4382)
16. Triple-layer flask (VWR, Cat #62407-082)
17. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #7346273)

Comment: To screen for compounds that activate the RGS4 protein, a HEK293 cell line which stably expresses M3R and inducibly expresses RGS4 is employed. RGS4 function is monitored by calcium flux with a commercially available Fluo4-AM dye. Compounds that do not show increase in the Fluo4 fluorescence in induced RGS4 expressed cells are considered as activator/potentiator hits. M3 receptor and other endogenous receptor activators/potentiators will be excluded through later counter-screening against non-induced parental cells.

External ID: SERCaMPGLuc-p1-antagonist

Protocol: PROTOCOL TABLES
SEQUENCE No. (1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE and DESCRIPTION.
1; Reagent; 5 uL; 1000 SH-SY5Y cells per wells
2; Time; 5 hour; 37C, 5% CO2
3; Compound; 23 nL; Control inhibitor / compound library
4; Time; 16 hour; 37C, 5% CO2
5; Reagent; 100 nM; Thapsigargin
6; Time; 4 hour; 37C, 5% CO2
7; Reagent; 1 uL; 0.5x coelenterazine
8; Detection; Luminescence; ViewLux imaging system

NOTES (numbers refer to sequence above)
1; SH-SY5Y human neuroblastoma cells stably expressing GLuc-SERCaMP (SH-SY5Y-GLuc-ASARTDL) cells were seeded in 1,536 well white tissue culture treated plates (Corning, Cat# 7464) in DMEM-high glucose-sodium pyruvate (ThermoFisher Scientific, Cat #10569) supplemented with 10% bovine growth serum (Hyclone), 10 U/ml penicillin (Gibco), 10 ug/ml streptomycin (Gibco), and 20 mM HEPES.
2; Assay plates were incubated for 5 hour at 37C in a humidified incubator containing 5% CO2.
3; qHTS libraries (23 nl, final concentrations of 1.53 uM, 7.67 uM, 38.3 uM) or controls (neutral control: DMSO, positive control: dantrolene) were added using a Kalypsis pin-tool Robotic System equipped with 1536 pinheads.
4; Cells were then incubated for 16 hours at 37C, 5% CO2.
5; Thapsigargin was added at 100 nM to deplete ER calcium stores.
6; Cells were incubated for 4 hour (37oC, 5% CO2)
7; Gaussia luciferase in the medium was measured by adding 1 ul of 0.5x coelenterazine (final concentration 0.07x) prepared in Gaussia Luciferase Glow Assay Buffer (Pierce), without addition of the Cell Lysis Buffer Reagent.
8; Luminescence was measured using a ViewLux high-432 throughput CCD imaging system (Perkin Elmer) equipped with clear filters. Compounds exhibiting inhibitory activity (defined as curve class -1.1, -1.2, -1.3, -1.4, -2.1, -2.2, -2.3, -2.4, -3) were identified by normalizing plate-wise to corresponding intra-plate controls (neutral control = Tg only; positive control (100% inhibition) = DMSO vehicle) with percent activity derived using in-house software (https://tripod.nih.gov/curvefit). The same controls were also used for the calculation of the Z' factor, a measure of assay quality control, as previously described (Zhang et al., 1999). For the initial validation of activity in the SERCaMP assays, hits from the primary screen were assayed again at 11-concentrations (1.3 nM - 76.6 uM). SH-SY5Y-GLuc-SERCaMP cells were assayed for ER Ca2+ depletion as outlined above.

Reference:
1. Zhang, J.H., Chung, T.D., and Oldenburg, K.R. (1999). A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4, 67-73.

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

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

External ID: CHEMBL659698

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: 2002
Volume: 45
Issue: 7
First Page: 1447
Last Page: 1459
DOI: 10.1021/jm010267o

Target ChEMBL ID: CHEMBL3571
ChEMBL Target Name: Cannabinoid CB1 receptor
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: CHEMBL5337074

Protocol: N/A

Comment: Journal: J Med Chem
Year: 2024
Volume: 67
Issue: 2
First Page: 1580
Last Page: 1610
DOI: 10.1021/acs.jmedchem.3c02148

Target ChEMBL ID: CHEMBL3571
ChEMBL Target Name: Cannabinoid CB1 receptor
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: CHEMBL4296184

Protocol: N/A

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

Target ChEMBL ID: CHEMBL352
ChEMBL Target Name: Staphylococcus aureus
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: CO-ADD Antimicrobial Screening

External ID: HMS1315

Protocol: Cybrid cells were washed with PBS, trypsinized, spun down at 300g for 5 minutes, and resuspended in 10 mLs of PBS. Cells were counted, and the appropriate amount of cells were added to a vial containing DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose, DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO, DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A, or DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose. Cells were then seeded in a Corning 3570 384-well plate (40 ul/well,1500 cells/well). Two replicates were prepared as described at the same time per library plate.

100 nl of compound was added to each well via pin transfer immediately after seeding. After the addition of compound, cells were incubated for 72 hours at 37 degrees C with 5% CO2.

Following the 72 hour incubation period, the 384 well plates were centrifuged for 1 minute at 1000 rpm. The media was aspirated using an aspiration wand and fresh media DMEM only, was added to the cells (40uL/ well) using a well-mate. Fifteen-microliters of Cell Titer Glow substrate was then added to each well using the well-mate. The plates were gently vortexed for 10 seconds and then centrifuged for 1 minute at 1000 rpm. The plates were then immediately read on the Envision instrument and the luminescence was detected for each individual well.

Positive control (strong): Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose
Positive control (weak): Cells seeded in DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A
Negative control: Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO

Comment: Data analysis method and criteria for scoring active compounds:

Z-scores were calculated for both replicates separately using the plate average and standard deviation of experimental well luminescence (Z = (x - mu)/sigma). Compounds were considered active if both replicate Z-scores >= 1.8. Activity scores were determined by scaling replicate average Z-scores from 0 (activity score = 0) to 4 (activity score = 100), with activity score > 45 being considered active. Z-scores < 0 were set to activity score = 0; Z-scores > 4 were set to activity score = 100 (100% activity).

External ID: CHEMBL4296186

Protocol: N/A

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

Target ChEMBL ID: CHEMBL350
ChEMBL Target Name: Klebsiella pneumoniae
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: CO-ADD Antimicrobial Screening

External ID: CHEMBL4296185

Protocol: N/A

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

Target ChEMBL ID: CHEMBL354
ChEMBL Target Name: Escherichia coli
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: CO-ADD Antimicrobial Screening

External ID: HMS979

Protocol: Cation-adjusted Mueller Hinton II broth supplemented with 0.005% Tween-80 was inoculated with a single colony of S. aureus RN4220. Following overnight incubation, the culture was diluted 1:100 into fresh medium, and incubation was continued until the bacterial suspension reached an optical density at 600 nm (OD600) of 0.6, corresponding to a bacterial density of 5 x 10E8 colony forming units (CFU)/mL. An aliquot of this culture was diluted 1:400 with fresh medium and stored on ice until use. Assay plates were prepared for compound transfer in quadruplicate to enable screening of compounds at two temperatures in duplicate. The plate layout was as follows: wells in columns 1-22 were loaded with culture medium at 25 microL/well; wells in column 23 contained the screening negative control (medium only); wells in column 24 contained the screening positive control (medium containing 25 microg/mL chloramphenicol). 300 nL of experimental compounds were transferred by stainless steel pin array from library plate to assay plates.

25 microL of the bacterial suspension was added to each assay well. Assay plates were incubated at 42 degrees C in humidified chambers (humidity > 85%). After 20 h, the OD600 was measured using a plate reader in absorbance mode.

Comment: Data analysis description:

Absorbance values at 42 degrees C and 30 degrees C for each replicate were normalized to positive and negative control plate average absorbance, and replicate normalized values were averaged to calculate an average relative absorbance at both temperatures. A substance was considered a temperature-dependent positive at 42 degrees C if average relative absorbance <= 50 and the difference between relative absorbance at 30 degrees C and 42 degrees C >= 20 (relative absorbance 30 degrees C - relative absorbance 42 degrees C >= 20). A substance was considered a temperature-independent positive if relative absorbance at both 30 degrees C and 42 degrees C < 10.

Activity scores were calculated using average relative absorbance at both temperatures. Average relative absorbance <= 0 was scored as 100 for activity; average relative absorbance >= 100 was scored as 0 for activityy. Average relative absorbance between 0 and 100 was subtracted from 100 to generate activity scores for intermediate values (i.e. average relative absorbance = 40 corresponds to an activity score of 60).

Note that since this is treated as a panel assay, the query for active compounds includes many that were considered active at both temperatures. The final Pubchem activity score is the activity score at 42 degrees C, and compounds scored as active (PUBCHEM_ACTIVITY_OUTCOME = 2) include both temperature-dependent and temperature-independent active substances.

External ID: IucA Pilot Assay Tocris Library

Protocol: A solution containing 55.6 mM HEPES pH 7.5, 0.11% Tween 20, 16.7 mM MgCl2, 55.6 mM hydroxylamine, 55.6 microM ATP, 55.6 microM citrate, and 0.28 U/mL IPP was dispensed (45 microL) into clear polystyrene microplates (Corning, Inc.) using a BioTek MicroFlo dispenser.

Next, 40 nL of test compounds (10 mM in DMSO, 8 microM final concentration) were transferred from deep-well blocks to the reaction solution using a stainless-steel pin tool operated by a robotic workstation (JANUS, PerkinElmer, Waltham, MA). The IucA-catalyzed reaction was initiated by adding 5 microL of 3 microM IucA in 25 mM HEPES, 75 mM NaCl, and 0.1 mM TCEP at pH 7.5.

The reactions were allowed to proceed for 30 min at room temperature before being quenched by dispensing (microFill, BioTek) 13 microL of MG developing solution, containing 1.0 mg/mL MG oxalate, 1.5% (w/v) ammonium molybdate, 0.15% (v/v) Tween 20, and 4.7 N sulfuric acid. After allowing the assay color to develop/stabilize for 30 min, the absorbance at 620 nm was measured (EnVision 2103 Multilabel Microplate Reader, PerkinElmer).

Average positive controls from 24 wells with no test compound and average negative controls from 8 wells with no enzyme were calculated. Dynamic range was calculated by difference between Avg Pos Control and Avg Neg Control. Percent inhibition was calculated by the ratio of (Avg. Pos. Ctrl - Sample OD) to Dynamic Range.

Comment: Protein Target is
IucA

EMB09144
574 aa
G057_19877
Klebsiella pneumoniae hvKP1

Active compounds were defined by <80% activity at 8 microM screening concentration.

External ID: CHEMBL4360723

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2019
Volume: 178
First Page: 571
Last Page: 588
DOI: 10.1016/j.ejmech.2019.05.037

Target ChEMBL ID: CHEMBL3571
ChEMBL Target Name: Cannabinoid CB1 receptor
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: HMS1482

Protocol: Prior to screening, cells were passaged in high glucose DMEM supplemented with 10% FBS, 1% P/S, 1 mM pyruvate, and HEPES. Media was changed every 1-2 days to prevent starvation and maintain cellular health.

On the day of screening, 384-well plates (Corning 3765) were filled with 30 uL of control cybrid cell suspension (33.3 cells/ul). Media for resuspension was DMEM supplemented with 10% FBS, 1% P/S, 1 mM pyruvate, and HEPES. Positive control columns were 23 and 24 for most plate setups. Initially, tunicamycin (15 nM) and galactose media (10 mM + 1 mM glucosamine) were used in columns 23 and 24, respectively. Over the course of the screen, galactose was substituted with Vidofludimus (11 uM). Next, 33 nL of each compound were pin-transferred to each plate in duplicate. Final assay well volume was 30 uL. Plates were stacked 2 high, covered with lids, and incubated at 37 degrees C for 2 days.

After 2 days of incubation, Hoescht (10 ul/well) was added to assay plates and fluorescence was read using the Acumen laser scanning cytometer. After, the bottom of plates were sealed with two layers of Brightmax Sealing Films. Then Promega NanoGlo Live cell reagent (20 ul/well) was added to each well and luminescence was read on a PerkinElmer EnVision plate reader after 5 minute of shaking.

Comment: Data analysis method and criteria for scoring active compounds:

Normalized luminescence values (luminescence / cell number) for each replicate were calculated (based on average of 2 luminescence reads per replicate). Z-scores were calculated using the plate average and standard deviation of experimental well normalized values. Each plate replicate was individually analyzed to measure reproducibility across plates. Z-scores were then averaged across replicates and a Z-score threshold of 1.96 was selected for positive hits, indicating increased mitochondrial supercomplex formation. Wells were excluded from further consideration based on low cell number (1 or both replicates with number of objects count < 1500). Activity scores were determined by scaling Z-scores from 0 (activity score = 0) to 4 (activity score = 100), with activity score > 50 being considered active. Z-scores < 0 were set to activity score = 0; Z-scores > 4 were set to activity score = 100 (100% activity).

External ID: CHEMBL4701646

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2020
Volume: 30
Issue: 22
First Page: 127501
Last Page: 127501
DOI: 10.1016/j.bmcl.2020.127501

Target ChEMBL ID: CHEMBL218
ChEMBL Target Name: Cannabinoid CB1 receptor
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

External ID: epac1-activator-v

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

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

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

External ID: PROCASPASE3_ACT_EPIABS_1536_1X%ACT PRUN

Protocol: Assay Overview:

The purpose of this assay is to identify compounds that activate procaspase 3 activity. This assay employs a procaspase 3 mutant enzyme, D9A/D28A/D175A (called PC-3 D3A) which is unable to autoproteolyze itself because its aspartic acid cleavage sites have been mutated to alanines. The mutant has a fully functional active site that can process the peptidic Ac-DEVD-pNA chromogenic substrate. Cleavage of substrate by procaspase 3 hydrolyzes the bond between the aspartic acid and p-nitroaniline, leading to release of yellow p-nitroaniline and an increase in well absorbance at 405 nm. In this assay, PC-3 D3A enzyme is pre-incubated with test compounds, followed by addition of substrate and measurement of well epi-absorbance. As designed, compounds that activate procaspase 3 activity will increase substrate hydrolysis, leading to an increase in well absorbance. Compounds are tested in singlicate at a nominal concentration of 8.5 uM.

Protocol Summary:

Prior to the start of the assay, 2.5 uL of zinc-free Assay Buffer (50 mM HEPES, 300 mM NaCl, pH 7.4, 0.01% Triton-X 100) containing 2 uM of PC-3 D3A protein were dispensed into 1536 microtiter plates. Next, 43 nL of test compound in DMSO or DMSO alone (0.8% final concentration) were added to the appropriate wells and incubated for 1 hour at 25 C.

The assay was started by dispensing 2.5 uL of 400 uM Ac-DEVD-pNA in Assay Buffer to all wells. Plates were centrifuged and after 2 hours of incubation at 25 C, epi-absorbance was read on the EnVision plate reader using a photometric filter set (excitation = 405 nm, emission = 450 nm) and a dichroic mirror with 425 nm cutoff. Fluorescence emission was read at 10 flashes per well at two time points (0 minutes and 120 minutes).

Prior to further calculations, the following formula was used to calculate absorbance:

Abs = ( -Log10( ( [ Raw2 ] / [ Mean Reference2 ] ) ) - ( -Log10 ( ( [ Raw1 ] ) / [ Mean Reference ] ) )

Where:

Raw1 is defined as the read at T0 minutes.
Raw2 is defined as the read at T120 minutes.
Mean Reference is defined as a mean of values from wells containing buffer only at T0.
Mean Reference2 is defined as a mean of values from wells containing buffer only at T120.

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:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.
High_Control is defined as wells containing DMSO and 5 uM PC-3 D3A.

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 100-9, and for inactive compounds 9-0.

List of Reagents:

Recombinant PC-3 D3A (procaspase 3 enzyme) (Assay Provider)
Assay Buffer (Assay Provider)
Chromogenic Substrate (Ac-DEVD-pNA) (Assay Provider)
1536 SWSN plates (Corning, part 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. 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 absorbance. 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: epac1-inhibitor-v

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

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

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

External ID: epac2-activator-v2

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

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

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

External ID: 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.

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.

External ID: CHEMBL4303805

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: VANDERBILT_HTS_GIRK2_MPD

Protocol: To screen for modulators of the G protein-gated inwardly-rectifying potassium channel subunit 2 (GIRK2) homomeric channels, HEK293 cells were engineered to overexpress GIRK2. These cells were screened using a high-throughput thallium-flux assay in a 384-well format. High-throughput thallium flux assays are based on three principles: (1) extracellular thallium can permeate through potassium channels into cells, (2) the fluorescent dye Thallos enters and stays in the cytoplasm of HEK293 cells, and (3) the fluorescence of Thallos increases dramatically when it interacts with thallium ions. Compounds that modulate the activity of an overexpressed channel, in this case GIRK2, are identified by the change in fluorescence that results from changes in thallium influx into cells through the overexpressed channel.

The screening protocol was conducted as follows. One hour prior to screening, cells were loaded with 1.5 micromolar (muM) Thallos in assay buffer (1x Hank's Balanced Salt Solution and 20 millimolar (mM) HEPES). The liquid handling and imaging was conducted using a kinetic-imaging plate reader, Panoptic (WaveFront Biosciences). Before screening, Thallos-containing buffer was removed from cells and replaced with 20 microliters (muL) per well of assay buffer. Compounds were dissolved in assay buffer at 20 muM. After 8 seconds of imaging, 20 muL of compound solution was added per well and allowed to incubate for 150 seconds. Next, 10 muL of a solution containing 2.0 mM thallium in Base Buffer (125 mM NaHCO3, 1 mM MgSO4, 1.8 mM CaSO4, 5mM Glucose, and 20 mM HEPES) was added to the wells. 30 seconds later, 12 muL of base buffer containing 2.0 mM thallium and 50 mM 2-methyl-2,4-pentanediol (MPD), a partially-activating concentration, was added to each well. MPD has is a previously-described GIRK2 channel activator. Data was collected for 60 seconds after this final addition. The positive control for this screen was 30 muM ivermectin, a natural product previously identified as a GIRK2 activator as part of a small-scale pilot screen. Microsoft Excel was utilized for data analysis. Data were normalized, F/Fo, on a well-to-well basis to account for differences in cell number. Compound activity was analyzed by comparing the sums of control-subtracted amplitudes of fluorescence intensity at 20 seconds after each thallium addition. The top 0.2% compounds corresponding to wells that demonstrated the largest increases in fluorescence were selected as "hits" for counter screening, as described below.

All 63,228 compounds screened using the method described above are listed in column OUTCOME_SCREEN_HITS. The 133 compounds selected as hits based on the above criteria are labelled with "100" in the ACTIVITY column. Fluorescent compounds were included in the inactive compounds and marked with "0" in the ACTIVITY column.

The 133 active compounds from column OUTCOME_SCREEN_HITS were tested for non-specific activity in untransfected HEK293 cells using assay conditions otherwise identical to the primary screen. Column OUTCOME_HEK_COUNTERSCREEN lists the 23 compounds that displayed an increase in fluorescence upon thallium addition with "100" in the ACTIVITY column.

The 110 compounds which were identified as hits in the primary screen but inactive when tested in untransfected HEK293 cells were tested for activity in GIRK2-overexpressing HEK293 cells that did not express NPY4 receptor. Column OUTCOME_GIRK2_COUNTERSCREEN lists the 44 compound that displayed an increase in fluorescence upon thallium addition with "100" in the ACTIVITY column.

The 44 active compounds from column OUTCOME_GIRK2_COUNTERSCREEN were tested at various concentrations to determine if compound activity was concentration dependent. Column OUTCOME_GIRK2_DOSE_RESPONSE provides the efficacy of these compounds; activity of all compounds at the provided concentrations was normalized to the activity of VU0537695 at 25 muM, which was the highest efficacy observed during this screening. For each compound concentration listed, the efficacy of that compound provided refers to the normalized, control-subtracted fluorescence intensity at 15 seconds after the addition of thallium. The 28 compounds that demonstrated >5% efficacy and concentration-dependent activity were labelled as "100" in the ACTIVITY column.

Finally, column PUBCHEM_ACTIVITY_OUTCOME combines all of the information from the above counterscreens together, indicating the 28 hits from this GIRK2 screen.

Comment:

External ID: CHEMBL4296188

Protocol: N/A

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

Target ChEMBL ID: CHEMBL614425
ChEMBL Target Name: Acinetobacter baumannii
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: CO-ADD Antimicrobial Screening