External ID: SNCA-p-activity-luciferase

Protocol: PROTOCOL TABLE
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION

1; Cells; 4 uL; Dispense 1500 HEK-293-SNCA-luc cells/well into Greiner 1536-well white / solid bottom tissue culture treated plate. The plate was covered with metal lids with gas-exchange holes.
2; Incubate; 24 hours; Incubate at 37C, 5% CO2, 95% RH.
3; Compounds; 23 nL; Compounds and controls were transferred via a Kalypsys Pin Tool (Wako USA) equipped with a 1536-slotted pin array. The plate was covered with metal lids with gas-exchange holes.
4; Incubate; 24 hours; Incubate at 37C, 5% CO2, 95% RH.
5; Dispense; 1 uL; Dispense Gly-Phe-7-amino-4-trifluoromethylcoumarin (GF-AFC, prepared at 125 uM in PBS) was added. The plate was covered with metal lids with gas-exchange holes.
6; Incubate; 30 min; Incubate at 37C, 5% CO2.
7; Detector; Fluorescence; Measure fluorescence with ViewLux microplate reader (PerkinElmer) equipped with 405/10 excitation and 540/25 emission filters.
8; Dispense; 3 uL; Dispense ONE-Glo (PerkinElmer) lucifase detection reagent was added to each well. Plates were covered with metal lids with gas-exchange holes.
9; Incubate; 15 min; Incubate at room temperature.
10; Detector; Luminescence; Measure luminescence with ViewLux microplate reader (PerkinElmer) equipped with clear filters.

NOTES (numbers refer to sequence above)
1; HEK-293-SNCA-luc were cultured and suspended in phenol-red free DMEM (4.5 g/L glucose, 25 mM HEPES, cat #21063 (Thermo)).
3; Compounds were added to the assay plate in an 11-point intra plate dose response, 1:3 titration in DMSO with a final concentration range of xxx - yyy uM. Vehicle-only plates, with DMSO being pin-transferred to every well, were inserted at the beginning of screening runs to confirm expected assay performance. Activity was normalized to wells containing medium only (-100% activity, full inhibition) and SNCA-luc cells treated with DMSO vehicle control (0% activity), contained on the same plate as test samples.
10; Signals were analyzed, and dose-response curves were fit using the Hill equation. Compounds in curve classes -1.1, -1.2, -2.1, -2.2 in the SNCA-luc assay were considered active. Compounds were eliminated from further consideration if also active (curve class -1.1, -1.2, -1.3, -1.4, -2.1, -2.2, -2.3, -2.4) in the GF-AFC cytotoxicity assay.

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: CHEMBL1640081

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: 2010
Volume: 53
Issue: 24
First Page: 8441
Last Page: 8460
DOI: 10.1021/jm100888d

Target ChEMBL ID: CHEMBL1641356
ChEMBL Target Name: Aldehyde oxidase
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: CHEMBL1789906

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: Science
Year: 2011
Volume: 334
Issue: 6061
First Page: 1372
Last Page: 1377
DOI: 10.1126/science.1211936

Target ChEMBL ID: CHEMBL612889
ChEMBL Target Name: Plasmodium yoelii
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL1789905

Protocol: N/A

Comment: Journal: Science
Year: 2011
Volume: 334
Issue: 6061
First Page: 1372
Last Page: 1377
DOI: 10.1126/science.1211936

Target ChEMBL ID: CHEMBL612889
ChEMBL Target Name: Plasmodium yoelii
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL3242317

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem. Lett.
Year: 2014
Volume: 24
Issue: 7
First Page: 1830
Last Page: 1838
DOI: 10.1016/j.bmcl.2014.02.006

Target ChEMBL ID: CHEMBL3879801
ChEMBL Target Name: NON-PROTEIN TARGET
ChEMBL Target Type: NON-MOLECULAR - Target has not been defined at a molecular level, only the non-molecular entity which is affected (e.g., organism, cell line etc)
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: HMS1625

Protocol: The fix-reporter plasmid was conjugated into this strain which the stably integrates into the chromosome using the mini-Tn7 system (PMID: 15908923). This plasmid consists of the fixK promoter, which is a target of the fixLJ pathway driving expression of GFP.
The day before screen an overnight culture of B. multivorans strain VC7102, with fix reporter, is set up in tryptic soy broth (TSB) at 37 degrees C with shaking. The day of the screen the overnight culture is diluted 1:100 in fresh tryptic soy broth.
The day of the screen, 30 microL of tryptic soy broth (TSB) is pipetted into columns 1-22 of a 384-well plate (Corning 3764) using the Combi multidrop dispenser. To column 24, 30 microL of TSB containing 78 microM of Benserazide TSB is added for a positive control. To column 23, 30 microL of TSB containing with DMSO (0.078% v/v in TSB) is added for a negative control. 300 nL of each compound are pin-transferred to each plate. For every compound plate 2 replicate assay plates are set up. 30 microL of the 1:100 diluted-B. multivorans strain VC7102 is added to each well using the Combi. Initial OD600 and GFP are measured using the PerkinElmer EnVision. Plates are stacked 5 high, covered with lids and incubated at 37 degrees C overnight (~18 h).
The following day, assay plates are read using a PerkinElmer EnVision (600 nm filter and GFP).
Positive control: Benserazide 39 microM in column 24 (No positive control was used for plates 2089 & 2090; Plates 2091-2093: columns 1 & 24)
Negative control: DMSO 300 nL/well in column 23 (For plates 2089 & 2090 negative controls were located in columns 2 & 24; plates 2091-2093: columns 2 & 23)

Comment: Analysis method:
For each compound well, the initial GFP value was subtracted from overnight GFP value to calculate the deltaGFP. The mean and standard deviation of the negative control wells deltaGFP (column 23) on both replicate plates was calculated. Wells with replicate average deltaGFP at least 3 standard deviations above the plate negative control deltaGFP average were scored as potential positives.

To determine activity scores, Z-scores were calculated for each replicate separately based on the replicate negative control deltaGFP plate average and standard deviation. The replicate average deltaGFP Z-scores were used for activity scores, with activity score of 100 set to Z-score = 4 and activity score = 0 (no activity) set to Z-score = 0. Z-scores > 4 were considered activity score = 100, and Z-scores < 0 were considered activity score = 0.

External ID: CHEMBL3878513

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: 2016
Volume: 124
First Page: 1081
Last Page: 1092
DOI: 10.1016/j.ejmech.2016.10.024

Target ChEMBL ID: CHEMBL3879801
ChEMBL Target Name: NON-PROTEIN TARGET
ChEMBL Target Type: NON-MOLECULAR - Target has not been defined at a molecular level, only the non-molecular entity which is affected (e.g., organism, cell line etc)
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL4622624

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: ACS Med Chem Lett
Year: 2020
Volume: 11
Issue: 8
First Page: 1653
Last Page: 1658
DOI: 10.1021/acsmedchemlett.0c00298

External ID: CHEMBL2210856

Protocol: N/A

Comment: Journal: Bioorg Med Chem Lett
Year: 2012
Volume: 22
Issue: 24
First Page: 7641
Last Page: 7646
DOI: 10.1016/j.bmcl.2012.10.010

Target ChEMBL ID: CHEMBL2189159
ChEMBL Target Name: Indoleamine 2,3-dioxygenase 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

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: CHEMBL1016905

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: 2008
Volume: 43
Issue: 12
First Page: 2840
Last Page: 2852
DOI: 10.1016/j.ejmech.2008.02.005

Target ChEMBL ID: CHEMBL364
ChEMBL Target Name: Plasmodium falciparum
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL5382778

Protocol: N/A

Comment: Journal: J Med Chem
Year: 2023
Volume: 66
Issue: 8
First Page: 5415
Last Page: 5426
DOI: 10.1021/acs.jmedchem.2c01663

Target ChEMBL ID: CHEMBL4883
ChEMBL Target Name: Urokinase plasminogen activator surface 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: CHEMBL765494

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1981
Volume: 24
Issue: 8
First Page: 1016
Last Page: 1018
DOI: 10.1021/jm00140a022

Target ChEMBL ID: CHEMBL612653
ChEMBL Target Name: Plasmodium berghei
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL765495

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1981
Volume: 24
Issue: 8
First Page: 1016
Last Page: 1018
DOI: 10.1021/jm00140a022

Target ChEMBL ID: CHEMBL612653
ChEMBL Target Name: Plasmodium berghei
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL5263088

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: 2017
Volume: 139
First Page: 982
Last Page: 1015
DOI: 10.1016/j.ejmech.2017.08.014

Target ChEMBL ID: CHEMBL5131
ChEMBL Target Name: Trypanothione 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: CHEMBL852081

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem. Lett.
Year: 2001
Volume: 11
Issue: 19
First Page: 2655
Last Page: 2658
DOI: 10.1016/s0960-894x(01)00528-5

External ID: CHEMBL766946

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: 1993
Volume: 3
Issue: 10
First Page: 2087
Last Page: 2092
DOI: 10.1016/S0960-894X(01)81022-2

Target ChEMBL ID: CHEMBL2096674
ChEMBL Target Name: Cyclooxygenase
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: CHEMBL1249739

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: Nat Chem Biol
Year: 2006
Volume: 2
Issue: 8
First Page: 415
Last Page: 416
DOI: 10.1038/nchembio806

Target ChEMBL ID: CHEMBL4651321
ChEMBL Target Name: Heme
ChEMBL Target Type: SMALL MOLECULE - Target is a small molecule such as an amino acid, sugar or metabolite)
Relationship Type: M - Molecular target other than protein assigned
Confidence: Target assigned is molecular non-protein target

External ID: CHEMBL635338

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1984
Volume: 27
Issue: 4
First Page: 450
Last Page: 465
DOI: 10.1021/jm00370a007

Target ChEMBL ID: CHEMBL345
ChEMBL Target Name: Nucleic Acid
ChEMBL Target Type: NUCLEIC-ACID - Target is DNA, RNA or PNA
Relationship Type: M - Molecular target other than protein assigned
Confidence: Target assigned is molecular non-protein target

External ID: ZIK159

Protocol: Assay Protocol Summary:

The medium for SNB-19 cells is composed of RPMI 1640 (ATCC, Cat.# 30-2001), 10% fetal bovine serum (FBS) (GE healthcare Life Sciences, Cat.# SH30071.03), and 1% Pen/Strep (Gibco, Cat.# 15140-122). A Caspase-Glo 3/7 assay kit (catalog number G8092; Promega, Madison, WI) was used to detect caspase-3 activity induced by Zika virus infection in human cells. The reagent mixture was reconstituted as described in the protocol from the manufacturer. Polystyrene tissue culture treated and PDL coated white plates were obtained from Greiner Bio-One (Monroe, NC). Cells were seeded in 384- or 1536-well assay plates and cultured at 37 C with 5% CO2 for 16 to 20 hours. The typical cell seeding density in the 1536-well plate assay is 250 cells/well in 3ul medium for SNB-19 cells in tissue culture treated plates. Compounds were added to cells and incubated for one hour before addition of ZIKV solution to cells (2 FFU/cell). After incubation at 37 C with 5% CO2 for 6 hours, the reagent mixture of Caspase-Glo 3/7 assay kit was added to each well, followed by incubation at room temperature for 30 minutes. The luminescence intensity of the assay plates was measured using a ViewLux plate reader (PerkinElmer). Data were normalized by using the cell-containing wells without ZIKV as a negative control (0% induction of caspase 3/7 activity) and wells containing ZIKV infected cells (Caspase-3 activity induced) as a positive control (100% induction of caspase 3 activity). The percentage inhibitions of the increased Caspase-3 activity by small molecule inhibitors were then calculated.

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: CF Folding

Protocol: A high throughput 384-well microplate based fluorescence assay was developed using HeLa cells engineered to express F508del-CFTR. Cells were maintained in cell culture media (High Glucose DMEM (Gibco) supplemented w/ 10% FBS (Gibco)). For the assay, cells were trypsinized (0.25% Trypsin-EDTA solution (Gibco)) and resuspended in assay media (cell culture media + 1% Pen/Strep (Gibco)) at a density of 320,000 cells per ml. Cells were dispensed to the assay plates (Corning 3712) in 25 ul using a Wellmate (Matrix/Thermo). Plates were incubated overnight at 37C, 5% CO2 and high humidity. The next day, compounds were prepared by making a dilution in assay media to a concentration of either 150 uM or 60 ug/ml (6x) depending on the library being screened. Then 5 ul of the diluted compound was transferred to the assay plate containing the cells. Assay plates were incubated for 24 hours 37C, 5% CO2 and high humidity. Following incubation with the compounds, relative cell number was determined by adding 3 ul of alamarBlue (TREK Diagnostics) and incubating the plates at 37C, 5% CO2 and high humidity, until cell control values reached ~4 million, as measured with a fluorescent intensity protocol on an Envision multimode plate reader (Perkin Elmer) (excitation 535 nm, emission 595 nm wavelengths) . Following the alamarBlue read, the media was removed and the cells were fixed with the addition of 13 ul of 4% paraformaldehyde in PBS (pH 7.4) and incubated for 10 minutes at room temperature. Plates were washed twice with PBS to remove the fixative. Cells were permeabilized with the addition of blocking solution (PBS, 0. 1% Triton X-100, 10 mg/ml BSA) and incubated at room temperature for 30 minutes. The blocking solution was removed and the primary antibody mixture was added in 13ul (Antibodies UNC570 and UNC596; CFF Therapeutics, Inc., Bedford, MA) at a 1/5000 dilution of each antibody in blocking solution. Plates were then incubated over night at 4oC. Primary antibody was removed by washing three times with 50 ul of wash solution (PBS, 0.1% Triton X-100) with at least 5 minutes between wash cycles. Secondary antibody (Alexa 488 anti-mouse IgG Invitrogen) was then added in a volume of 13 ul (1/1000 dilution in blocking solution) and incubated for two hours at room temperature in the dark. To remove excess secondary antibody, plates were washed three times with 50 ul of wash solution and one time with 50 ul of PBS. Plates were read from the bottom using a fluorescent intensity protocol on an Envision multimode plate reader (485 nm excitation and 535 nm emission).
To minimize the disruption of the cell monolayer during the numerous washes, the following process was used. The removal of media or other reagents for the wells of the microtiter plate was done using a Biomek FX (Beckman, Fullerton CA) with a 384 tip multichannel head. Aspiration was done at a slow rate following the liquid level with the tip positioned in the top right corner of the well. All solution additions, starting with the fixative, were done with a Matrix Wellmate. Again to avoid disturbing the cell monolayer, the plate offset was adjusted so that the liquid dispensed to the left wall of the well rather than directly on the cell monolayer.
For the screen, approximately ten thousand compounds from a mix of FDA and diverse libraries (MicroSource Pharmacon-1600 (FDA), Enzo (FDA), Selleck (FDA) and Enamine) were tested at a final concentration 25 uM (Enzo, Selleck & MS Pharmakon) or 10 ug/ml (Enamine 30K diversity set). The proteasome inhibitor ALLN was used as the positive control (50 uM) and 100 uM Hyamine was used as a viability control on all microtiter plates. All wells including the cell controls contained 0.5% DMSO. In-plate controls were used to normalize the data and results were reported as fold increase above the cell control and normalized to relative cell number derived from the alamarBlue data.
Ninety six compounds identified in the single dose screen were evaluated further in dose response. Compound high dose was twice the screening concentration and a ten point series of two fold dilutions were done for each compound. DMSO concentration was 1% for in all wells. Compounds were evaluated in both the immunostain assay and in the cell viability assay.

Data Analysis: Thirty two control wells containing cells only, 24 wells containing Hyamine and 8 wells containing ALLN were included on each assay plate and used to calculate Z' value for each plate and to normalize the data on a per plate basis. Data were analyzed using the IDBS Activity Base software. Results were expressed as Fold Increase (FI) for the immunostain assay and cell viability (V) for the alamarBlue assay. Final data was reported as Normalized fold increase (NFI). Data was analyzed as follows FI=DataValue/Median cell control, V= (DataValue-median Hyamine control)/(median cell control -median Hyamine control) and NFI=FI/V.

For the immunostain dose response assay, IC50 values were calculated for active compounds by plotting the FI for each concentration, and using a 4 parameter Levenburg-Marquardt algorithm with the minimum limit set at 0. Similarly, EC50 values were calculated for the cell viability dose response assay, plotting the % Viability at each concentration and also using a 4 parameter Levenburg-Marquardt algorithm with the minimum limit set at 0 and the maximum limit set at 100.

Outcome- From the primary screen, compounds selected as Active were those that met one of the following two criteria: (1) Fold Induction >/= 1.5, Cell Viability >/= 50% and the ratio of Fold Induction to Cell Viability >/= 1.5; or (2) Fold Induction >/= 1, Cell Viability >/= 50% and the ratio of Fold Induction to Cell Viability >/=1.75. While 101 compounds met this criteria, 96 were ultimately selected for confirmatory screening. For the dose response screen, compounds were labeled as "Active" if they showed at least 8 fold increase over the ALLN control at any concentration.

Comment: Possible Artifacts: Possible artifacts in this assay include, but are not limited to, compounds that are fluorescent, absorb at the excitation or emission wavelengths, or non-specifically upregulate protein expression

External ID: CHEMBL766130

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: 1993
Volume: 3
Issue: 4
First Page: 711
Last Page: 716
DOI: 10.1016/S0960-894X(01)81260-9

Target ChEMBL ID: CHEMBL2111358
ChEMBL Target Name: Prostaglandin G/H synthase (cyclooxygenase)
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: CHEMBL1039052

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: 2009
Volume: 52
Issue: 23
First Page: 7503
Last Page: 7511
DOI: 10.1021/jm900920x

Target ChEMBL ID: CHEMBL3879801
ChEMBL Target Name: NON-PROTEIN TARGET
ChEMBL Target Type: NON-MOLECULAR - Target has not been defined at a molecular level, only the non-molecular entity which is affected (e.g., organism, cell line etc)
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL908049

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem. Lett.
Year: 2006
Volume: 16
Issue: 17
First Page: 4515
Last Page: 4518
DOI: 10.1016/j.bmcl.2006.06.025

Target ChEMBL ID: CHEMBL3880
ChEMBL Target Name: Heat shock protein HSP 90-alpha
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: CHEMBL936363

Protocol: N/A

Comment: Journal: Bioorg. Med. Chem.
Year: 2008
Volume: 16
Issue: 7
First Page: 3759
Last Page: 3772
DOI: 10.1016/j.bmc.2008.01.053

Target ChEMBL ID: CHEMBL3253
ChEMBL Target Name: Serum albumin
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: CHEMBL5634745

Protocol: N/A

Comment: Journal: Eur J Med Chem
Year: 2023
Volume: 259
First Page: 115702
Last Page: 115702
DOI: 10.1016/j.ejmech.2023.115702

Target ChEMBL ID: CHEMBL360
ChEMBL Target Name: Mycobacterium tuberculosis
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL4193896

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: ACS Med Chem Lett
Year: 2017
Volume: 8
Issue: 8
First Page: 886
Last Page: 891
DOI: 10.1021/acsmedchemlett.7b00230

External ID: CHEMBL1249757

Protocol: N/A

Comment: Journal: Nat Chem Biol
Year: 2006
Volume: 2
Issue: 8
First Page: 415
Last Page: 416
DOI: 10.1038/nchembio806

Target ChEMBL ID: CHEMBL4651321
ChEMBL Target Name: Heme
ChEMBL Target Type: SMALL MOLECULE - Target is a small molecule such as an amino acid, sugar or metabolite)
Relationship Type: M - Molecular target other than protein assigned
Confidence: Target assigned is molecular non-protein target

External ID: CHEMBL1040691

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: Proc Natl Acad Sci U S A
Year: 2008
Volume: 105
Issue: 26
First Page: 9059
Last Page: 9064
DOI: 10.1073/pnas.0802982105

Target ChEMBL ID: CHEMBL364
ChEMBL Target Name: Plasmodium falciparum
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: Novartis Malaria Screening

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: CHEMBL1040694

Protocol: N/A

Comment: Journal: Proc Natl Acad Sci U S A
Year: 2008
Volume: 105
Issue: 26
First Page: 9059
Last Page: 9064
DOI: 10.1073/pnas.0802982105

Data Source: Novartis Malaria Screening

External ID: CHEMBL1040692

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: Proc Natl Acad Sci U S A
Year: 2008
Volume: 105
Issue: 26
First Page: 9059
Last Page: 9064
DOI: 10.1073/pnas.0802982105

Target ChEMBL ID: CHEMBL364
ChEMBL Target Name: Plasmodium falciparum
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: Novartis Malaria Screening

External ID: CHEMBL1040693

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: Proc Natl Acad Sci U S A
Year: 2008
Volume: 105
Issue: 26
First Page: 9059
Last Page: 9064
DOI: 10.1073/pnas.0802982105

Target ChEMBL ID: CHEMBL614039
ChEMBL Target Name: Huh-7
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: Novartis Malaria Screening

External ID: CHEMBL1217000

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: 2010
Volume: 18
Issue: 6
First Page: 2225
Last Page: 2231
DOI: 10.1016/j.bmc.2010.01.068

Target ChEMBL ID: CHEMBL364
ChEMBL Target Name: Plasmodium falciparum
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL958548

Protocol: N/A

Comment: Journal: J. Nat. Prod.
Year: 1997
Volume: 60
Issue: 11
First Page: 1158
Last Page: 1160
DOI: 10.1021/np970221r

Target ChEMBL ID: CHEMBL5584
ChEMBL Target Name: Phospholipase A2, acidic
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: CHEMBL3301363

Protocol: N/A

Comment: Data Source: AstraZeneca DMPK/physicochemical

External ID: CHEMBL3301365

Protocol: N/A

Comment: Data Source: AstraZeneca DMPK/physicochemical

External ID: CHEMBL767139

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1996
Volume: 39
Issue: 26
First Page: 5183
Last Page: 5191
DOI: 10.1021/jm960437a

Target ChEMBL ID: CHEMBL3474
ChEMBL Target Name: Phospholipase A2 group IIA
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: HMS1303

Protocol: Prior to screening, lyophilized FITC-GIV peptide (a.a. 1671-1701) is dissolved in 100% DMSO to a concentration of 0.5 mM, then further diluted with water to a final concentration of 50 microM. One stock was prepared in batch for the entire screen and aliquoted. All protein stocks were stored at -80C. Thawing of aliquots on the day of the assay was performed rapidly at room temperature.

Prior to transfer of screening compounds, experimental wells of 384-well assay plates were pre-filled with 37.8 nM of FITC-GIV peptide (for 25 nM final concentration) in a 10 microL volume of assay buffer (50 mM Tris [pH 7.4], 100 mM NaCl, 10 mM MgCl2, 5 mM EDTA, 0.4% NP-40, 30 microM GDP, 1 mM DTT). Control wells were pre-filled with 37.8 nM of FITC-GIV peptide in 10 microL of assay buffer containing either 1% DMSO (vehicle negative control) or 45.3 microM AlCl3+15.1 mM NaF (AlF4- positive control). Each plate contained 16 negative control and 16 positive control wells. Plates were centrifuged at 1000xg for 2 minutes to eliminate bubbling on the surface. Following, 100 nL of experimental compound was pin-transferred into individual wells for each of two replicate assay plates (A&B). After pin-transfer, 5 microL of 3 microM His-tagged rat Galphai3 purified from E. coli (for 1 microM final concentration) was added to each well, and assay plates were shaken at low speed for 5 seconds. Final assay volume was 15 microL. Plates were centrifuged at 1000xg for 2 minutes to eliminate bubbling on the surface and shaken for 10 seconds prior to an incubation of 90 minutes at room temperature. Plates were protected from prolonged light exposure. All manipulations were performed at room temperature.

Assay plates were read with a PerkinElmer Wallac EnVision Multilabel 2103 plate reader using the fluorescent polarization measurement technology with a D505fp/D535 dual mirror and a 480 nm excitation filter. 535 nm emission filters were used for P and S channel reads at 11 mm measurement height with 30 flashes and detector gains of 319 and 563. G-factor 0.96.

Negative control: DMSO
Positive control: Aluminum tetrafluoride (AlF4-, composite of NaF and AlCl3)

Comment: Calculated result values and scoring of active compounds:

Z-score: Indicates the number of standard deviations an experimental condition is from the mean. It is calculated based on plate average (u) and standard deviation (s) of experimental well fluorescence polarization (FP) measurements for each replicate. Z-score (z) is defined as the quotient of the mean of the experimental well population subtracted from an individual well's FP (x), divided by the standard deviation within the experimental wells; z = (x-u)/s.

Normalized percent control: calculated by subtracting plate average positive control FP from experimental well FP, dividing by the difference between plate average negative and positive control FP, and multiplying by 100.

STD: the number of standard deviations from the plate average negative control FP for each replicate experimental well

Compound wells with FP values exceeding +5 standard deviations from the negative control sample average were flagged as potential aggregators or fluorescence quenchers due to their high polarization signal (HighPol) and were excluded from further consideration. Activity scores were calculated by averaging the replicate normalized percent of control values and subtracting from 100 (score based on single replicate if data for other replicate was excluded for any reason). Result values < 0 were set to 0 (no activity) and > 100 were set to 100 (100% activity). Compounds with activity score >= 15 were considered active.

External ID: CHEMBL947292

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: 2008
Volume: 16
Issue: 14
First Page: 6737
Last Page: 6746
DOI: 10.1016/j.bmc.2008.05.060

Target ChEMBL ID: CHEMBL3879801
ChEMBL Target Name: NON-PROTEIN TARGET
ChEMBL Target Type: NON-MOLECULAR - Target has not been defined at a molecular level, only the non-molecular entity which is affected (e.g., organism, cell line etc)
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL767146

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1995
Volume: 38
Issue: 14
First Page: 2794
Last Page: 2797
DOI: 10.1021/jm00014a032

Target ChEMBL ID: CHEMBL3474
ChEMBL Target Name: Phospholipase A2 group IIA
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: CHEMBL632931

Protocol: N/A

Comment: Journal: J Med Chem
Year: 2002
Volume: 45
Issue: 13
First Page: 2867
Last Page: 2876
DOI: 10.1021/jm0200409

External ID: CHEMBL948326

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: 2008
Volume: 16
Issue: 14
First Page: 6737
Last Page: 6746
DOI: 10.1016/j.bmc.2008.05.060

Target ChEMBL ID: CHEMBL3879801
ChEMBL Target Name: NON-PROTEIN TARGET
ChEMBL Target Type: NON-MOLECULAR - Target has not been defined at a molecular level, only the non-molecular entity which is affected (e.g., organism, cell line etc)
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL948328

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: 2008
Volume: 16
Issue: 14
First Page: 6737
Last Page: 6746
DOI: 10.1016/j.bmc.2008.05.060

Target ChEMBL ID: CHEMBL3879801
ChEMBL Target Name: NON-PROTEIN TARGET
ChEMBL Target Type: NON-MOLECULAR - Target has not been defined at a molecular level, only the non-molecular entity which is affected (e.g., organism, cell line etc)
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL948327

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: 2008
Volume: 16
Issue: 14
First Page: 6737
Last Page: 6746
DOI: 10.1016/j.bmc.2008.05.060

Target ChEMBL ID: CHEMBL3879801
ChEMBL Target Name: NON-PROTEIN TARGET
ChEMBL Target Type: NON-MOLECULAR - Target has not been defined at a molecular level, only the non-molecular entity which is affected (e.g., organism, cell line etc)
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular