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: 20130627FPNS3INTERFERECB

Protocol: Assay Overview:

The purpose of this biochemical assay is to determine the ability of compounds in a ChemBridge compound library to interfere with the fluorescence signal of a Cy5-labeled substrate oligonucliotide of the hepatitis C non-structural protein 3 helicase (NS3h). In this biochemical assay, the fluorescence of a single strand of DNA with a 5' Cy5 fluorophore (Cy5-dT15) incubated with NS3h and test compound is monitored.

Protocol Summary:

Assays were performed in 20 uL in 384-well black small-volume microplates. All assays contained 5 nM Cy5-dT15 DNA (5'- Cy5 TT TTT TTT TTT TTT T -3'), 15 nM NS3h, 25 mM MOPS, pH 7.5, 1.25 mM MgCl2, 0.0025 mg/ml BSA, 0.005% (v/v) Tween 20, and 0.025 mM DTT. Compounds dissolved in DMSO were added at final concentration of 0.1mM and a final DMSO concentration of 1% (v/v). Cy5 fluorescence was read. Interference was calculated according to the equation below.

Interference = Fc / F[-]

Where:

Fc is the fluorescence in the presence of the compound
F[-] is the average fluorescence negative controls

Any compound that exhibited an interference value greater than 0.8 or less than 1.2 was considered inactive. Any compound that exhibited an interference value greater than or equal to 1.2 was considered active (a fluorescent enhancer) in the assay and will not be pursued further. Any compound that exhibited an interference value less than or equal to 0.8 was considered active (a fluorescent quencher) in the assay and will not be pursued further.

List of Reagents:

Cy5-dT15 (Integrated DNA Technologies Inc, custom synthesized)
dT20 (Integrated DNA Technologies Inc, custom synthesized)
MOPS (Fisher Scientific, part BP308-100)
Magnesium Chloride (Fisher Scientific, part BP214-500)
384-well plates (Greiner Bio-One, black, part 784076)

Comment: The assay was performed and submitted by the Frick Lab at the University of Wisconsin-Milwaukee. The compounds tested in this assay were from a ChemBridge compound library.

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: 20130624FPNS3DACB

Protocol: Assay Overview:

The purpose of this biochemical assay is to determine the ability of compounds in a ChemBridge compound library to displace the hepatitis C non-structural protein 3 helicase (NS3h) from its substrate oligonucleotide. In this biochemical assay, the fluorescence polarization of a single strand of DNA with a 5' Cy5 fluorophore (Cy5-dT15) incubated with NS3h and test compound is monitored. Fluorescence polarization increases when NS3h binds the substrate. As designed, compounds that interfere with binding decrease the fluorescence polarization.

Protocol Summary:

Assays were performed in 20 uL in 384-well black small-volume microplates. All assays contained 5 nM Cy5-dT15 DNA (5'- Cy5 TT TTT TTT TTT TTT T -3'), 15 nM NS3h, 25 mM MOPS, pH 7.5, 1.25 mM MgCl2, 0.0025 mg/ml BSA, 0.005% (v/v) Tween 20, and 0.025 mM DTT. Compounds dissolved in DMSO were added at final concentration of 0.1mM and a final DMSO concentration of 1% (v/v). Cy5 fluorescence polarization was read using a G-factor calculated from a well lacking NS3h or test compounds. Percent inhibition was calculated according to the equation below.

%_Inhibition = 100 - ( ( Fc - F[+]) / (F[-] - F[+] ) ) * 100

Where:

Fc is the fluorescence polarization in the presence of the compound
F[-] is the average fluorescence polarization of no enzyme negative controls
F[+] is the average fluorescence polarization of positive controls (200 nM dT20).

Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter. Any compound that exhibited greater percent inhibition than the cutoff parameter was considered active.

List of Reagents:

Cy5-dT15 (Integrated DNA Technologies Inc, custom synthesized)
dT20 (Integrated DNA Technologies Inc, custom synthesized)
MOPS (Fisher Scientific, part BP308-100)
Magnesium Chloride (Fisher Scientific, part BP214-500)
384-well plates (Greiner Bio-One, black, part 784076)

Comment: The assay was performed and submitted by the Frick Lab at the University of Wisconsin-Milwaukee. The compounds tested in this assay were from a ChemBridge Library set.

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%.