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: LDHA

Protocol: The assay was optimized to 1536-well plates. Briefly, 3 muL of human lactate dehydrogenase 5 (#A38558H, Meridian Life Science, Inc., Memphis, TN) in LDH assay buffer (200 mM Tris HCl pH 7.4, 100 microM EDTA and 0.01% Tween-20) was added to a black solid bottom 1536-well assay plate (Greiner Bio-One) using a BioRAPTR FRD dispenser (Beckman Coulter, Brea, CA). A 1536-well pintool dispenser outfitted with 20 nL pins (Wako Automation, San Diego, CA) was used to transfer 23 nL of DMSO-solubilized compound (both library and vehicle controls) to each 1536-well assay plate. Following compound transfer, 1 muL of substrate solution containing NADH and sodium pyruvate (Sigma-Aldrich, St. Louis, MO) in LDH assay buffer was dispensed via BioRAPTR FRD to initiate the reaction. Final concentrations in the 4 microL reaction volume were 2 nM LDHA enzyme, 0.06 mM NADH and 0.2 mM sodium pyruvate. Following a 5 minute incubation period at room temperature, 1 muL of detection reagent (C. kluyveri diaphorase (Sigma-Aldrich) and resazurin sodium salt (Sigma-Aldrich) in LDH assay buffer) was added to a total volume of 5 muL. Final concentrations of detection reagents were 0.133 mg/mL diaphorase and 37 muM resazurin. Plates were immediately transferred to a ViewLux microplate imager (PerkinElmer, Waltham, MA), and any resulting resorufin fluorescence was measured (ex540, em590 nm) at 0 and 20 minutes. Fluorescence was normalized using enzyme-free and DMSO-treated control wells on each plate.

Comment: PubChem score indicates % inhibition at 229 uM or 114 uM of the compound (Max_response). Active compounds with Max_response greater than -100%, PUBCHEM_ACTIVITY_SCORE is 100. Active compounds with Max_response between -80% and -100%, PUBCHEM_ACTIVITY_SCORE is 80. Active compounds with Max_response between -60% and -80%, PUBCHEM_ACTIVITY_SCORE is 60. Active compounds with Max_response between -30% and -60%, PUBCHEM_ACTIVITY_SCORE is 40. Inconclusive Compounds with Max_response between -25% and -30% have PUBCHEM_ACTIVITY_SCORE 20. For all inactive compounds with Max_response greater than -25%, PUBCHEM_ACTIVITY_SCORE is 0

External ID: ST2_IL33_Inhibitors_Primary_Screening_77700

Protocol: High-throughput screening was performed at Indiana University screening facility (Indiana University, Indianapolis, IN).

In summary, a mixture of ST2 and IL-33 was prepared by adding 0.30 microL of 14.1 microM recombinant human ST2-Fc Chimera and 0.30 microL of 55.25 biotinylated recombinant human IL-33 to 1.4 mL of assay buffer. In the HTS, each well of a 384-well Proxi-plate was first blocked by 100 microL of assay buffer for 1 h at room temperature. After removing the blocking buffer, 20 microL of ST2/IL-33 mixture was pipetted into each well and incubated at room temperature for 1 h followed by addition of compounds at 17 microM (0.8 microL) to each well and incubated at room temperature for 1 h. Ten microliters of 60 microg/mL anti-6xHis-conjugated AlphaLISA acceptor beads were added to each well and incubated at room temperature for 1 h before addition of 10 microL of 60 microg/mL of streptavidin-labeled AlphaLISA donor beads for incubation at room temperature for 30 min. Incubation of acceptor and receptor beads was conducted in darkness. The well with 3 nM ST2 alone was used as a negative control, whereas the ST2/IL-33 mixture with a human ST2 antibody added at 0.45 ng/microL was used as the positive control. Plates were read using the Envision Plate Reader. The buffer used in the assay contains 14.37 mL PBS, 30 microL Tween 20, and 600 microL of 5% bovine serum albumin in PBS.

Comment: PubChem active indicates >= 30% inhibition of ST2/IL-33 at 17 uM of the compound. Inconclusive: >=10% and < 30% inhibition.

External ID: CHEMBL1634179

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 2010
Volume: 53
Issue: 23
First Page: 8362
Last Page: 8367
DOI: 10.1021/jm100993z

Target ChEMBL ID: CHEMBL4624
ChEMBL Target Name: Fibonectin-binding protein C
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: stopgo-p2-SytraCBC-dual-FF

Protocol: Reagents:
StopGo2A HEK-NPG-Luc cells and other necessary media and controls were provided by Dr. Atkins's lab. Compound PTC124 was used as a control. Dose response was assessed using a 1:2 dilution of control compound, with the top dose of 50uM final concentration. Background signal was normalized to wells treated with DMSO only.

Assay Protocol:
In brief, four microliters (2400 cells/well) of StopGo2A HEK-NPG-Luc cells were plated and incubated overnight at 37C. Twenty-three nanoliters of compounds and control were added to the wells using a 1536 array Kalypsys pintool workstation. After an over night incubation (at 37C) with compounds / control, 2.5 uL of Dual/Glo Firefly reagent was added. Firefly luciferase signal was obtained using the PerkinElmer ViewLux plate reader after 20min of room temperature incubation. Dual/Glo Renilla Lucifrase reagents were then added followed by additional 20 min room temperature incubation. Finally, the renilla luciferase signal was obtained using the PerkinElmer ViewLux reader.

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: 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: stopgo-p2-SytraCBC-dual-Ren

Protocol: Reagents:
StopGo2A HEK-NPG-Luc cells and other necessary media and controls were provided by Dr. Atkins's lab. Compound PTC124 was used as a control. Dose response was assessed using a 1:2 dilution of control compound, with the top dose of 50uM final concentration. Background signal was normalized to wells treated with DMSO only.

Assay Protocol:
In brief, four microliters (2400 cells/well) of StopGo2A HEK-NPG-Luc cells were plated and incubated overnight at 37C. Twenty-three nanoliters of compounds and control were added to the wells using a 1536 array Kalypsys pintool workstation. After an overnight incubation (at 37C) with compounds / control, 2.5 uL of Dual/Glo Firefly reagent was added. Firefly luciferase signal was obtained using the PerkinElmer ViewLux plate reader after 20min of room temperature incubation. Dual/Glo Renilla Lucifrase reagents were then added followed by additional 20 min room temperature incubation. Finally, the renilla luciferase signal was obtained using the PerkinElmer ViewLux reader.

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: CHEMBL1634181

Protocol: N/A

Comment: Journal: J Med Chem
Year: 2010
Volume: 53
Issue: 23
First Page: 8362
Last Page: 8367
DOI: 10.1021/jm100993z

Target ChEMBL ID: CHEMBL613088
ChEMBL Target Name: Mycolicibacterium smegmatis
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

External ID: CHEMBL1634182

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 2010
Volume: 53
Issue: 23
First Page: 8362
Last Page: 8367
DOI: 10.1021/jm100993z

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: HSPH_Screening_CFS_002

Protocol: Preparation of assay plates and seeding cells:
Polyacrylamide based gel substrates were miniaturized in glass bottom 96-well plates. In the first method, each 96-well plate was treated with NaOH (6N in water) for 1hr followed by silane solution ((3-aminopropyl)trimethoxysilane, 10% in water) for an additional 1hr. Then, glass surfaces were treated with glutaraldehyde (0.25% in PBS) for 30min and further washed and dried. Acrylamide gels (5.5% acrylamide, 0.076% bisacrylamide, Young's modulus = 1.8kPa, thickness = 200 microm) were cast in each well using a custom-made gel caster. The gel surfaces were functionalized using sulfo-SANPAH (sulfosuccinimidyl-6-[4 -azido-2 -nitrophenylamino]hexanoate, 0.2mg/mL), coated with green beads (0.2microm sulfate microspheres, Invitrogen, 0.002% in water), coated with bovine collagen I (40microg/mL in PBS) and were stored at 4C (Fig. 1A) for more than 1 day. After washing off collagen solution, primary human airway smooth muscle cells were seeded in each well (10,000 cells/well). 1 day after seeding, media was replaced with serum-deprivation media and cells were kept in serum free media for 2 days.

Measurements of contractile forces using Fourier-transform traction microscopy:
On the day of screening, 96-well plate was mounted upon a motorized stage within a temperature controlled chamber and imaged using an inverted microscope (DMI 6000B, Leica Inc.). In each well, images were obtained in quick succession: one phase contrast image of cells and a fluorescent images of beads. The image set was obtained before plating cells (reference), immediately prior to adding drugs (baseline), and 1 hr after drug addition (treatment). By comparing fluorescent images obtained during baseline or treatment with the corresponding image from reference, we computed the cell-exerted displacement field using particle image velocimetry. From the displacement field, we computed the contractile force (per unit area) using Fourier-transform traction microscopy modified to the case of cell monolayers. This modified approach takes into consideration effects of finite gel thickness as well as force imbalances associated with the microscope field of view as we described previously. From each force map, we computed the root mean squared value to represent the averaged contractile force. Drug effects were quantified as the 'force response ratio' (FRR), namely, the contractile force before versus after drug addition.

Small molecule library and pooling:
ChemBridge Diverset was screened with pooling 8 compounds together.
Primary measurements of the screening are FRR (force response ratio). 1 means no change in average contractile force and less than 1 means the reduction of average contractile force after drug treatment.

Comment: Among the mixtures having FRR less than 1, 12 most effective mixtures were chosen as positives and deconvoluted to test individually. Finally, 9 single compounds were found effective.

External ID: CHEMBL1634183

Protocol: N/A

Comment: Journal: J. Med. Chem.
Year: 2010
Volume: 53
Issue: 23
First Page: 8362
Last Page: 8367
DOI: 10.1021/jm100993z

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