External ID: FATTTLab-Algae-Lipid

Protocol: Cells were pre-grown to mid-log phase in TAP media. For primary screen, cells were seeded at low density of 500,000 / well in 384-well plate. Compound in DMSO was added to each well at a final concentration of 10 micromolar and cells were allowed to grow for 72 hours. At the end of assay, Nile Red (30 uM f.c.) was added and plates were incubated at 37 C for 60 mins in dark. Fluorescence intensity was measured. Readouts were reported as normalized fold change in the intensity of treated versus control.

Comment: Reference:
1. Wase, N., Tu, B., Black, P. N. & DiRusso, C. C. Phenotypic screening identifies Brefeldin A/Ascotoxin as an inducer of lipid storage in the algae Chlamydomonas reinhardtii. Algal Research 11, 74-84 (2015).

2. Identification and metabolite profiling of chemical activators of lipid accumulation in green algae
#Nishikant Wase, Boqiang Tu, James W Allen, Paul N Black, Concetta C DiRusso
#Plant Physiology Jun 2017, pp.00433.2017; DOI: 10.1104/pp.17.00433
#http://www.plantphysiol.org/content/early/2017/06/26/pp.17.00433

Patent:
DiRusso, C., & Wase, N. (2016). Compounds for Increasing Lipid Synthesis and Storage. United States#NUtech Ventures (Lincoln, NE, US) http://www.freepatentsonline.com/y2016/0312253.html

For additional Information please contact:

Prof. Concetta C. DiRusso
Department of Biochemistry
University of Nebraska-Lincoln
cdirusso2@unl.edu

Nishikant Wase, PhD
Department of Biochemistry
University of Nebraska-Lincoln
nishikant.wase@gmail.com

External ID: CHOL_MOT

Protocol: Vibrio cholerae motility assay; Soft agar motility assay Methods
Fifty nl of test compound was dispensed to a dry assay plate with an Echo550. Then 50ul of LB containing 0.3% agar was added to the plates with a Matrix/Thermo Wellmate. The soft agar containing assay plates were allowed to solidify at room temperature for at least 4 hours. Bacterial inoculation was done with an Echo 555 which was used to transfer 2.5 nl of bacterial culture, containing approximately 20 bacteria, to the soft agar plate. The 2.5 nl drop of bacterial culture was delivered to the corner of the well and was accomplished by adjusting the well offset for the destination plate on the Echo. Plates were incubated at 30C for 14 hours then read using absorbance at 615nm on a Perkin Elmer Envision. The read location in the well was offset diagonally to allow maximum distance between the inoculation site and the read location. The primary read was absorbance at 615 to monitor bacterial growth and motility from the inoculation site to the read location. To differentiate between non-motile and non-viable, alamarBlue was added following the absorbance read. Plates were incubated for 60 minutes at 30C and then fluorescence intensity was determined by reading with an excitation wavelength of 535 and an emission wavelength of 590nm using a top read and a well center location. Non-motile viable colonies convert the alamarBlue while non-viable wells remained blue allowing the discrimination between antimicrobial compounds and motility inhibitors.

Comment: Possible artifacts in the V. cholerae motility assay include, but are not limited to, compounds that auto fluoresce (false positive for viability read) and compounds that absorb in the 500-600 nm range (false negative), or precipitate.
Outcome: Compounds that inhibited motility (OD615 assay) but that did not inhibit bacterial viability (alamarBlue assay) were identified as actives. A total of 18 compounds were ordered screened in the confirmatory (dose response assay) at concentrations ranging from 50 to 0.1 uM. Two compounds showed IC50s of less than 12uM in the motility inhibition assay and were inactive in the alamarBlue bacterial viability inhibition assay. Two additional compounds had a selectivity index of greater than 3.5, were also considered "Active". Compound that were active in the primary screen that were not tested in the confirmatory set were labeled with an Outcome of "Inconclusive".
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score.
Compounds in the primary screen are scored on a scale of 0-40 based on the difference % inhibition of the motility and growth inhibition results. In the interests of time, the compounds were selected on first run values. The second primary screen replicate was used for statistical analysis of the reproducibility of the primary assay.

External ID: CHEMBL3232407

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 1979
Volume: 22
Issue: 7
First Page: 816
Last Page: 823
DOI: 10.1021/jm00193a013

Target ChEMBL ID: CHEMBL376
ChEMBL Target Name: Rattus norvegicus
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: TargetID_659_CEMA

Protocol: Black, standard capacity streptavidin-coated 384-well plates (Pierce 15407) were first washed with 50 L of phosphate buffer (100 mM, pH 7.0; PB7) three times using a Biotek 405 ELX plate washer. Subsequently, 5 L of biotinylated pre-miRNA substrate (500 nM final) was dispensed into the plate using a Multidrop Combi Reagent Dispenser (Thermo Scientific). Plates were then centrifuged for 1 min at 1,000 RPM (223 g), sealed with plate tape, and incubated overnight at 4 C. The following morning, plates were washed three times with 50 L of PB7, followed by the addition of 5 L of Dicer digest buffer (20 mM Tris, 12 mM NaCl, 2.5 mM MgCl2, 1 mM fresh DTT, and 4.5% DMSO) and centrifugation. Compounds (50 nL of 5 mM DMSO stock, 25 M final) were then added into the sample wells using a Sciclone (Caliper) liquid handler with V&P pintool; the same volume of DMSO was added to the control wells. The plates were incubated at 25 C for 15 min before addition of 5 L of digest buffer containing 217 g/nL Dicer (108 g/mL Dicer, 5% glycerol and 0.01% Triton X-100 final, excess with respect to pre-miRNA). For the positive control wells, digest buffer without Dicer was added. The plates were centrifuged again and resealed before being placed in a 37 C incubator for 5 h. After Dicer cleavage, plates were washed three times with 50 L of PB7. mTet-HRP in PB7 (10 L, 750 nM final) was then dispensed into each well. The plates were subsequently centrifuged, sealed, and incubated at 25 C for 2 h. Plates were then washed three times with 50 L of wash buffer (2 mM imidazole, 260 mM NaCl, 0.5 mM EDTA, 0.1% Tween-20, pH 7.0), followed by washing three additional times with 50 L of PB7. Finally, SuperSignal West Pico (25 L; Pierce) was added, the plates were incubated at 25 C for 5 min, and chemiluminescence signal was detected using a PHERAstar plate reader using LUM plus module (BMG Labtech).

Comment: The activity outcome is based on a Z-score (number of standard deviations from the negative control mean) of 3 or higher on at least 50% times that the sample was screened.

For instance, if the sample was screened in n=4 runs, it would be considered active only if it had a Z-score of 3 or above in at least 2 runs.

External ID: MCF7_OneDose

Protocol: NCI-60 Human Tumor Cell Lines Screen was performed using the standard one-dose NCI protocol, which is described in more detail at https://dtp.cancer.gov/discovery_development/nci-60/default.htm.

Compounds with GIPRCNT values below 10 were considered active. Activity score was based on GIPRCNT using the following formula:
max(-GIPRCNT/2 + 50, 0)
Score is 0 for GIPRCNT values of 100 or above, 50 for GIPRCNT values of 0, and 100 for GIPRCNT values of -100.

Comment: These data are a subset of the data from the NCI human tumor cell line screen. Compounds are identified by the NCI National Service Center (NSC) number, which is contained in the
PUBCHEM_EXT_DATASOURCE_REGID field. In the NCI cell line identification system, MCF7 is panel number 5, cell number 1. PANELCODE or PANELNAME identify the NCI-60 cell panel. PANELNBR does NOT identify the NCI-60 panel.

EXPID is the NCI internal tracking number for the experiment identifier, and PREFIX = 'S' for the NSCs submitted through the NCI Chemical Agents repository.

Substance concentration unit is indicated in the data table, using the following abbreviations: M = molar (M), u = micrograms/milliliter (microg/mL), V = volumetric fraction.

M_GIPRCNT represents the mean growth percent for that experiment, and N_GIPRCNT indicates the number of values for that NSC within an experiment, while STDEV_GIPRCNT is the standard deviation. Most NSCs are tested once per experiment, resulting in EXP_COUNT = 1.

External ID: MScreen:TargetID_600

Protocol: C-terminally 6xHis tagged CDK2/Cyclin A complex and N-terminally Flag tagged CDC25B C473S (372-566) were expressed and purified. Proteins were incubated together at a final concentration of 125 nM each for 1 hr prior to incubation with compound for 1 hr, followed by addition of anti-6xHis europium cryptate donor beads (Cisbio) and anti-Flag XL-665 acceptor beads (Cisbio) at a final dilution of 1:350 for 1 hr. 20mM potassium fluoride was added 10 minute prior to plate reading. Assay reagents were dispensed using a multidrop liquid dispenser (Thermo Scientific) onto uncoated, black, low-volume, 384-well plates (Corning). Assay plates were quantified using an Envision plate reader (Perkin-Elmer) with excitation of the europium crytate donor at 337 nm wavelength and emission of the donor at 620 nm and emission of the XL-665 acceptor at 665 nm in 18 uL volumes. Assays were performed in a buffer containing 50 mM Tris (pH 7.5), 50 mM NaCl, 10mM MgCl2, 1mM TCEP, with addition of and 1mM ATP, 0.05% BSA, and 0.05% Tween-20 immediately prior to the start of the assay.

Comment: The activity outcome is based on a Z-score (number of standard deviations from the negative control mean) of 3 or higher on at least 50% times that the sample was screened.
For instance, if the sample was screened in n=4 runs, it would be considered active only if it had a Z-score of 3 or above in at least 2 runs.

This screen was funded by NIH grant number: R01CA181185

External ID: IGROV1_OneDose

Protocol: NCI-60 Human Tumor Cell Lines Screen was performed using the standard one-dose NCI protocol, which is described in more detail at https://dtp.cancer.gov/discovery_development/nci-60/default.htm.

Compounds with GIPRCNT values below 10 were considered active. Activity score was based on GIPRCNT using the following formula:
max(-GIPRCNT/2 + 50, 0)
Score is 0 for GIPRCNT values of 100 or above, 50 for GIPRCNT values of 0, and 100 for GIPRCNT values of -100.

Comment: These data are a subset of the data from the NCI human tumor cell line screen. Compounds are identified by the NCI National Service Center (NSC) number, which is contained in the
PUBCHEM_EXT_DATASOURCE_REGID field. In the NCI cell line identification system, IGROV1 is panel number 6, cell number 10. PANELCODE or PANELNAME identify the NCI-60 cell panel. PANELNBR does NOT identify the NCI-60 panel.

EXPID is the NCI internal tracking number for the experiment identifier, and PREFIX = 'S' for the NSCs submitted through the NCI Chemical Agents repository.

Substance concentration unit is indicated in the data table, using the following abbreviations: M = molar (M), u = micrograms/milliliter (microg/mL), V = volumetric fraction.

M_GIPRCNT represents the mean growth percent for that experiment, and N_GIPRCNT indicates the number of values for that NSC within an experiment, while STDEV_GIPRCNT is the standard deviation. Most NSCs are tested once per experiment, resulting in EXP_COUNT = 1.

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%.