External ID: CHEMBL4649948

Protocol: N/A

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

Data Source: Gates Foundation Compound Collection

External ID: CHEMBL4649949

Protocol: N/A

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

Data Source: Gates Foundation Compound Collection

External ID: CHEMBL4649954

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: CHEMBL4662925
ChEMBL Target Name: Histidine--tRNA ligase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous single protein target assigned

Data Source: Gates Foundation Compound Collection

External ID: CHEMBL4649972

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: CHEMBL4662931
ChEMBL Target Name: ClpP1P2
ChEMBL Target Type: PROTEIN COMPLEX - Target is a defined protein complex, consisting of multiple subunits
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous protein complex subunits assigned

Data Source: Gates Foundation Compound Collection

External ID: CHEMBL3988443

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL4105741
ChEMBL Target Name: Methionyl-tRNA synthetase, putative
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous single protein target assigned

Data Source: Gates Foundation Compound Collection

External ID: CHEMBL4649941

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: CHEMBL4662928
ChEMBL Target Name: Coenzyme A biosynthesis bifunctional protein CoaBC
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous single protein target assigned

Data Source: Gates Foundation Compound Collection

External ID: CHEMBL4649973

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: CHEMBL4649981
ChEMBL Target Name: Wolbachia pipientis
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: Gates Foundation Compound Collection

External ID: VANDERBILT_HTS_GIRK2_HPP

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 and the neuropeptide Y receptor type 4 (NPY4). The purpose of NPY4 overexpression was to promote modest activation (~30% activity) of GIRK2 channels upon NPY4 activation with human pancreatic polypeptide (hPP). 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 and 3.5 nanomolar (nM) hPP 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 1 muM hPP, a maximally-activating concentration, was added to each well. Data was collected for 60 seconds after this final addition. The positive control for this screen was 16.6 muM eprinomectin, 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 0.5% compounds corresponding to wells that demonstrated the largest increases in fluorescence were selected as "hits" for counter screening, as described below.

All 34,131 compounds screened using the method described above are listed in column OUTCOME_SCREEN_HITS. The 167 compounds selected as hits based on the above criteria are labelled ACTIVE in this column. Fluorescent compounds were included in the inactive compounds and marked as INACTIVE.

The 167 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 39 compounds that displayed an increase in fluorescence upon thallium addition, labelled with ACTIVE.

The 128 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 8 compound that displayed an increase in fluorescence upon thallium addition, labelled with ACTIVE.

The 8 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 VU0529331 at 37.5 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. Compounds that demonstrated >5% efficacy and concentration-dependent activity were labelled as ACTIVE.

Finally, column Outcome combines all of the information from the above counterscreens together, indicating the 6 hits from this GIRK2 screen.

Comment:

External ID: CHEMBL4513217

Protocol: N/A

Comment: Journal: ACS Infect Dis
Year: 2020
Volume: 6
Issue: 4
First Page: 613
Last Page: 628
DOI: 10.1021/acsinfecdis.9b00482

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

Data Source: Winzeler Lab Plasmodium Screening

External ID: CHEMBL4513219

Protocol: N/A

Comment: Journal: ACS Infect Dis
Year: 2020
Volume: 6
Issue: 4
First Page: 613
Last Page: 628
DOI: 10.1021/acsinfecdis.9b00482

Target ChEMBL ID: CHEMBL395
ChEMBL Target Name: HepG2
ChEMBL Target Type: CELL-LINE - Target is a specific cell-line
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: Winzeler Lab Plasmodium Screening

External ID: CHEMBL4513218

Protocol: N/A

Comment: Journal: ACS Infect Dis
Year: 2020
Volume: 6
Issue: 4
First Page: 613
Last Page: 628
DOI: 10.1021/acsinfecdis.9b00482

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

Data Source: Winzeler Lab Plasmodium Screening

External ID: CHEMBL4649965

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: CHEMBL4105939
ChEMBL Target Name: Polyketide synthase Pks13
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous single protein target assigned

Data Source: Gates Foundation Compound Collection

External ID: CHEMBL4513221

Protocol: N/A

Comment: Journal: ACS Infect Dis
Year: 2020
Volume: 6
Issue: 4
First Page: 613
Last Page: 628
DOI: 10.1021/acsinfecdis.9b00482

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: Winzeler Lab Plasmodium Screening

External ID: CHEMBL4513220

Protocol: N/A

Comment: Journal: ACS Infect Dis
Year: 2020
Volume: 6
Issue: 4
First Page: 613
Last Page: 628
DOI: 10.1021/acsinfecdis.9b00482

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: Winzeler Lab Plasmodium Screening

External ID: CHEMBL3507681

Protocol: N/A

Comment: Data Source: Gates Foundation Compound Collection

External ID: KUHTS-Muma KU-CaM-Htt INH-01

Protocol: 1; Dispense 45 nl compounds (10 mM stocks) using ECHO 555 to Alpha 384 well assay plates. Dispense 45 nl DMSO to control columns 1 and 2 of 384 well plates.
2; Incubate 5 ul of 6XHis-mHTT (Final, 13 nM) with the compounds for 40 mins at room temperature in buffer containing 10 mM Tris.HCl pH 7.4, 1 mM calcium chloride, 150 mM sodium chloride, 0.1% BSA and 20% glycerol.
3; Dispense 5 ul of 6XGST-CaM (Final 13 nM) in buffer A.
4; Incubation; 1 hour (dark at 25C)
5; Dispense 20 ul of Nickel chelate acceptor beads (Final, 20 ugs/ml) and Glutathione donor beads (Final, 30 ugs/ml). Incubate for 2h, room temperature.
6; Detector: Perkin Elmer Enspire, Alphascreen Module (Excitation 680nm/Emission 570nm).

NOTES (numbers refer to Sequence numbers above)
1. Alphascreen bead incubations were performed in green light, TiterTek setting 400rpm.
2. All incubation and addition steps were followed by mixing and centrifugation at 400g,1 min.
3. The percent inhibition for each compound was calculated as follows:
100- [100 *((Test Compound-Median Low Control) / (Median High Control - Median Low Control))]

Where:
Test_Compound is defined as wells containing His mHTT + GST CaM in the presence of test compound

High_Control is defined as wells containing His mHTT + GST CaM and DMSO.

Low_Control is defined as wells containing His mHTT and DMSO.

Comment: All percent inhibition data reported were normalized to high and low controls on a per-plate basis. The results of primary screening data include compounds contributing to assay signal interference, interference with tags binding to Alpha beads, chelators, process related artifacts etc.. The actives were defined as compounds that inhibited Alphascreen reads to greater than 50%.

External ID: CHEMBL4649957

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL4662922
ChEMBL Target Name: Tryptophan--tRNA ligase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: H - Homologous protein target assigned
Confidence: Homologous single protein target assigned

Data Source: Gates Foundation Compound Collection

External ID: CHEMBL4649962

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: CHEMBL4662927
ChEMBL Target Name: Histidine--tRNA ligase
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

Data Source: Gates Foundation Compound Collection

External ID: WaveGuideAssay:441

Protocol: For initial screening, TetON human embryonic kidney cells (HEK293) expressing GluA2 and stargazin, gamma-2, were plated in 384-well BD purecoat amine-coated plates at 16k cells/well. Cells were induced with 8 ug/mL doxycycline in the presence of 30 uM NBQX and incubated O/N at 37deg in 5% CO2. They were washed with Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES, pH 7.4 and FLIPR VSD dye solution was added and incubated on the cells at room temperature for 45 minutes. The cell plate was loaded into the Hamamatsu Functional Drug Screening System 6000 (FDSS), 10 seconds of background were collected before addition of compound at 30 uM final concentration. Data collection continued at 1Hz sampling and experimentally determined EC50 glutamate stimulus was added at 300 seconds. Controls of 1 mM glutamate (maximal effect) and NBQX (antagonist) were used to calculate a Z' score per plate.

Data were processed using the Vanderbilt screening analysis platform, WaveGuide. Data were normalized by dividing the raw fluorescence for all time points by an average of the baseline fluorescence. For each well, 4 values were calculated as follows:

1. CMPDslope was the slope of the normalized value within the timeframe 11-18 seconds,
2. CMPDmaxmin was the difference in maximal and minimal normalized value within the timeframe 8-100 seconds
3. GLUslope was the slope of the normalized value within the timeframe 310-315 seconds, and
4. GLUmaxmin was the difference in maximal and minimal normalized value within the timeframe 301-380 seconds.

Compounds were marked as hits where any of the above calculated results for a compound differed by at least three standard deviations from the mean for that calculation across all compounds in the plate.

Compounds denoted as 'hits' were given the score of '100' and outcome of 'active'. All other compounds were denoted as 'inactive' and given a score of '0'.

Comment: