External ID: CHEMBL1266185

Protocol: N/A

Comment: Journal: Nat Chem Biol
Year: 2007
Volume: 3
Issue: 5
First Page: 268
Last Page: 273
DOI: 10.1038/nchembio873

External ID: HMS1262

Protocol: NEC is stored at -80 degrees at a concentration of 15mg/ml in single use aliquots.

On the day of the screen, 20ul of purified NEC is aliquoted using a Multidrop Combi reagent dispenser into 384 well plates (Corning 3824). 100nl of compound dissolved in DMSO was transferred to each well of the assay plated via pin transfer. The plates (NEC + compound) are incubated at room temperature for 3 hours. Acceptor and donor reagents (CisBio 620/665 pair) are combined then added to each well at 5 microL volumes at a concentration of 8 nM and 80nM respectively. The plates are spun at 1k rpm for 1 min and incubated overnight at 4 degrees, then for one hour the subsequent day at room temperature.

Flourescent measurements are read on the Envision 1 plate reader at ICCB-L. The raw data consists of two fluorescence readings - at 665 nm and 620 nm for the acceptor and donor respectively.

Comment: Data analysis:
The raw data consists of two fluorescence readings - at 665 and 620 nm for the acceptor and donor respectively. The data is processed as a ratio of the emission from the acceptor over the donor (homogeneous time resolved fluorescence ratio). Normalized percent inhibition (NPI) for all experimental wells is calculated based on plate averages for negative and positive control HTRF ratio. Positives are scored as any ratio with a 50% or greater inhibition as compared with the positive control (i.e. NEC + Untagged UL50). To be considered a hit, both replicates need to score as positive. Activity scores are derived from NPI, with 100 = 100% inhibition (> 100% set to 100) and 0 = no inhibition (< 0% set to 0). Note that some compounds with NPI <50% (activity scores < 50) are classified as potential hits based on additional criteria (typically by selecting wells with low ratios compared to other experimental wells on the plate).

External ID: PolI100

Protocol: Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol iota in columns 1, 2, and 5-48) will be dispensed into 1,536-well black solid-bottomed plate. Compounds (23 nL) will be transferred via Kalypsys pin tool equipped with 1536-pin array. The plates will then be incubated for 15 min at room temperature, and 1 muL substrate (50 nM final concentration) will be added to start the reaction and kinetically read twice at 0 min and 10 min on the Viewlux reader.

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: PolE100

Protocol: Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol eta in columns 1, 2, and 5-48) were dispensed into a 1,536-well black solid-bottomed plate. Compounds (23 nL) were transferred via Kalypsys pin tool equipped with 1536-pin array. The plates were then incubated for 15 min at room temperature, and 1 muL substrate (50 nM final concentration) was added to start the reaction and kinetically read twice at 0 min and 10 min on the Viewlux reader

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: TDP1640

Protocol: Buffer: 1X PBS, pH 7.4, 80 mM KCl, 0.05% Tween-20

Reagents/Controls:
[1] 3 ul of buffer is dispensed in columns 3 & 4 as negative control (no enzyme and maximum signal),
[2] 3 ul of enzyme (1 nM final) is dispensed in columns 1,2, 5-48,
[3] 1 ul of substrate (15 nM final) is dispensed throughout the plate,
[4] 1 ul of AS donor/acceptor bead mix (10 ug/ml final) is dispensed throughout the plate

Assay steps:
3 ul of Tdp1 enzyme is dispensed to 1536-well Kalypsys black solid bottom plates. Compounds and controls (23 nl) are transferred via Kalypsys Pin Tool. The plates are incubated for 15 min at room temperature, and then 1 ul of substrate is added to start the reaction. After 5 min incubation at room temperature, 1 ul of AS donor/acceptor bead mix is added and the plates are further incubated for 10 min at room temperature. The detection is then performed on a PerkinElmer Envision reader.

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: 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: HERG01

Protocol: NCGC Assay Protocol Summary:

HERG assay (FluxORTM thallium flux assay) was initially developed by Invitrogen/Molecular Probes, and then miniaturized into 1536-well plate in a homogeneous format by NCGC. This assay measures the activity of potassium channel using thallium dye (FluxOR) flux as surrogate measurement for potassium into the cells with a FDSS-7000 kinetic plate reader (Hamamatsu Corp., Hamamatsu City, Japan). The hERG ion channel is transduced into mammalian cells (U2OS) using a baculovirus (Bacmam) construct harboring the hERG K+ ion channel. So far we screened LOPAC1280 library (Sigma), the NTP collection of 1408 compounds, and the NCGC Pharmaceutical Collection (NPC), in which many well defined HERG blockers are present. The rank order potencies of many of these compounds are similar to that of other HERG assays (membrane potential, Rb+ flux, patch clamp, etc). This quick and homogeneous assay is also found to be sensitive, specific, and robust.

Using the FluxORTM thallium flux assay, the activity of potassium channel using thallium dye (FluxOR) flux as surrogate measurement for potassium into the cells was measured in the U2OS cell line transduced with hERG K+ ion channel using a baculovirus (Bacmam) using Opti-MEM medium (Invitrogen) containing 2% fetal calf serum (FCS, HyCone) following loading buffer addition, compound treatment for around 10 minutes and finally adding stimulation buffer. The assay was performed in black clear Kalypsys 1536-well plates. In the screen, Astemizole was used as positive controls. Library compounds were measured for their ability to cause hERG channel blockage in the cell line, as reflected by a decrease in fluorescence intensity, in a concentration-dependent manner. Data were normalized to the controls for basal activity (DMSO only) and 100% inhibition (5uM Astemizole). AC50 values were determined from concentration-response data modeled with the standard Hill equation.

qHTS protocol for hERG-U2OS cellular assay

[Step] [Parameter] [Value] [Description]

1. Day 1: Replace medium in 70-80% confluent T225 flask with 2.5 mL of hERG-BacMam virus plus 12.5 mL of phosphate buffered saline (PBS) (corresponding roughly to a multiplicity of infection ratio of 100 virus particles/cell)
2. Incubation: 4 hrs @ room Temperature in Dark
3. Reagent; Remove virus; wash once with 25 ml DPBS
4. Reagent; 35 ml culture medium
5. Incubation; 37oC overnight
6. Day2: Reagent; 3 uL; 2000 U2OS cells/well
7. Time; 4 hr; 37oC incubation
8. Loading buffer; 1 uL; 0.7X;
9. Incubation: 1hr @ RT in Dark.
10. Compounds; 23 nL; 0.59 nM to 92 uM
11. Controls; 23 nL; Astemizole 1.4 nM to 92 uM
12. Time; 10min; 37oC incubation
13. Read fluorescence Intensity on FDSS for 10 Sec with 1 sec interval
14. Reagent; 1 uL; stimulation buffer
15. Read fluorescence Intensity on FDSS for 2 min with 1 sec interval
16. Detection; Fluorescence Intensity; FDSS

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: HMS1315

Protocol: Cybrid cells were washed with PBS, trypsinized, spun down at 300g for 5 minutes, and resuspended in 10 mLs of PBS. Cells were counted, and the appropriate amount of cells were added to a vial containing DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose, DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO, DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A, or DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose. Cells were then seeded in a Corning 3570 384-well plate (40 ul/well,1500 cells/well). Two replicates were prepared as described at the same time per library plate.

100 nl of compound was added to each well via pin transfer immediately after seeding. After the addition of compound, cells were incubated for 72 hours at 37 degrees C with 5% CO2.

Following the 72 hour incubation period, the 384 well plates were centrifuged for 1 minute at 1000 rpm. The media was aspirated using an aspiration wand and fresh media DMEM only, was added to the cells (40uL/ well) using a well-mate. Fifteen-microliters of Cell Titer Glow substrate was then added to each well using the well-mate. The plates were gently vortexed for 10 seconds and then centrifuged for 1 minute at 1000 rpm. The plates were then immediately read on the Envision instrument and the luminescence was detected for each individual well.

Positive control (strong): Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 1.25mM Glucose
Positive control (weak): Cells seeded in DMEM No Glucose + 2% FBS + 1% P/S + 10mM Galactose + 1uM I-BET GSK 525762A
Negative control: Cells seeded in DMEM (No Glucose) + 2%FBS + 1% P/S + 10mM Galactose + 0.3% DMSO

Comment: Data analysis method and criteria for scoring active compounds:

Z-scores were calculated for both replicates separately using the plate average and standard deviation of experimental well luminescence (Z = (x - mu)/sigma). Compounds were considered active if both replicate Z-scores >= 1.8. Activity scores were determined by scaling replicate average Z-scores from 0 (activity score = 0) to 4 (activity score = 100), with activity score > 45 being considered active. Z-scores < 0 were set to activity score = 0; Z-scores > 4 were set to activity score = 100 (100% activity).

External ID: ZIK097

Protocol: Assay Protocol Summary:

The medium for hNPCs consists of DMEM/F12, N2 supplement (ThermoFisher, Cat.# 17502048), NEAA (ThermoFisher, Cat. # 11140050), 2 ug/ml heparin, 2 uM cyclopamine, and B27 (ThermoFisher, Cat. # 17504044). 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 350 cells/well in 3 ul medium for hNPCs 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: TRXR100

Protocol: Assay protocol: 2 uL of reagents (buffer in column 4 as negative control and 90 nM rTrxR1 in columns 1-3 and 5-48) were dispensed into Greiner black solid-bottom 1,536-well assay plates, followed by 1 uL of NADPH (400 uM final concentration) to each well. The plates were centrifuged at 1000 rpm for 15 seconds and subsequently incubated for 5 min at room temperature (~22 deg C) to allow for rTrxR1 reduction. Compounds (23 nL) were then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). In addition, a duplicate 2-fold serial dilution of the control compounds auranofin, a known gold-based TrxR1 inhibitor, and juglone (5-hydroxy-1,4-naphthoquinone), a natural TrxR1 substrate, were pin-transferred to columns 2 and 3, respectively. After incubation for 15 min at room temperature (~22 deg C), 1 uL of selenite (400 uM final concentration) were dispensed to each well. The plate was immediately transferred to a ViewLux high-throughput CCD imager (PerkinElmer), wherein kinetic measurements of NADPH fluorescence (Ex 340 nm, Em 450 nm) were acquired (8 minute kinetic read, see Table 1). Read 1 was utilized to assess the capacity of a compound to serve as an rTrxR1 substrate, i.e. a decrease in NADPH fluorescence compared to the no-compound background is an indication of a substrate behavior for that particular compound. For inhibitory activity of a compound, delta values, computed as the difference in fluorescence intensity between the first and last reads of an 8-minute time kinetic window, were used. All reagents were diluted in an assay buffer consisting of 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 0.01% Tween-20.

Throughout the screen, reagent bottle and all liquid lines were made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a 'double-pinning' step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the columns 5-48, were inserted uniformly at the beginning and the end of each library in order to monitor and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity over time. Screening data were corrected, normalized, and concentration-effect relationships were derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts).

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: DDV296

Protocol: Four microliters of culture medium (RPMI 1640 with 0.5% w/v Albumax (GIBCO), 24 mM sodium bicarbonate and 10 ug/mL gentamycin) were dispensed by a Multi-drop Combi into white solid 1536-well plates (Grenier) and 23 nL compound was added by a pin tool. Four microliters of infected erythrocytes (2% hematocrit, 0.1% parasitemia final concentration) in culture medium were dispensed and the plates incubated for 96 hours at 37 C in 5% CO2. Two microliters of luciferase detection reagent was added and luminescence was detected by a ViewLux (PerkinElmer) reader

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: 20130523FPNS3DALOPAC

Protocol: Assay Overview:

The purpose of this biochemical assay is to determine the ability of compounds in Sigma-Aldrich's Library of Pharmacologically Active Compounds (LOPAC1280) 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 Sigma-Aldrich's LOPAC-1280 set.

External ID: MH081217, Chlamydomonas reinhardtii Gravitaxis Assay to Identify Small Molecule Inhibitors of Cilia.

Protocol: Detailed Protocol for Chlamydomonas reinhardtii Algae Gravitaxis Assay
Materials:
a. 96-well U-bottom plates (Costar 3798)
b. Adhesive seal film
c. 2M Tris-acetate (pH 7.0)
d. Phosphate buffer (pH 7.0)
e. Nutrient stock (contains NH4Cl, MgSO4, CaCl2)
f. Hunters Trace Elements (supplied by Dr. Wallace Marshalls lab)

To make one liter of liquid TAP media:
10 mL 2M Tris-acetate (pH 7.0) + 10 mL Phosphate buffer (pH 7.0) + 10 mL nutrient stock + 10 mL Hutner's trace elements (10x dilution), autoclave well.

Tris-Acetate Stock:
242 g Tris base
Dissolve in 600 mls water while titrating to pH 7.0 with glacial acetic acid.
Bring to 1 liter

Nutrient Stock:
NH4Cl 40 g
MgSO4.7H2O 10 g
CaCl2.2H2O 5 g
Bring to 1 liter.

Phosphate buffer
K2HPO4 10.8 g
KH2PO4 5.6 g
Bring to 1 liter

Hutners Trace Elements:
A) Dissolve 50g of acid free EDTA in 250 ml of ddH2O. Heat to dissolve.
B) Dissolve the following, one by one, in order, heating to approximately 100 C in 550 ml ddH2O.
BO3H3 11.40 g
ZnSO4,7H2O 22.00 g
MnCl2,4H2O 5.06 g
FeSO4,7H2O 4.99 g
CoCl2,6H2O 1.61 g
CuSO4,5H2O 1.57 g
Mo7O24(NH4)6,4H2O
(Ammonium Molybdate) 1.1 g

Mix the two solutions (A & B) together. The resulting solution should be blue-green. Heat to 100 C. Cool slightly, but don't let the temperature drop below 80-90 C. Adjust pH to 6.5 to 6.8 with 20% KOH (approximately 83 ml). Don't let the temperature drop below 70 C until after the pH is adjusted. Make up to 1L and let stand in a 2L Erlenmeyer flask, stoppered loosely. The color should change from green to purple over a period of days. Remove the rust-coloured precipitate by filtering, with suction, through 3 layers of Whatman number 1 filter paper in a Buchner funnel. Repeat until no more precipitate is seen on the filter paper. Store in a brown bottle at 4C.
Protocol:
1. Grow cells (WT cc124 test cells and mutant bld1 control cells) to OD600 ~ 0.5 in Erlenmeyer flasks (100mL media in a 250 ml flask) covered with aluminum foil and shaken in a rotary shaker. In order to allow sufficient illumination, a fluorescent light bulb (GE Soft While, Home Depot) is placed above the floor shaker and suspended from a shelf, with a distance of 1 foot between the top of the flasks and the lights. All growth is done ambient temperature (21C).
2. Check OD 600 nm on M5 plate reader after 3-4 days, and dilute algae to OD 0.1 in TAP media.
3. 5 uL of compounds are transferred to the U-bottomed assay plates (columns 2-11) using the Evolution-P3 liquid handler equipped with a 96-well transfer head.
4. 96-well U-bottom plates (column 2-11, plates: Costar 3798) are then seeded with 145 uL of WT cc-124 cells into using the Evolution-P3 liquid handler equipped with a 96-well transfer head.
5. WT cc-124 and MUT bld1 are seeded into the plate columns 1 and column 12 with 1.0 % DMSO (final), as min and max controls.
6. Plates are sealed with an adhesive transparent seal, and placed on the bench top for 2 days at ambient temperature with overhead fluorescent lights placed to provide even illumination.
7. When cells in the control wells are visible (i.e. small green circle are seen in the cc-124 control wells and green color is seen throughout the wells in the bld1 control plate) it is time to read the plates on the Flexstation 3 or M5 microtiter plate reader at OD 600nm.

Comment: Chlamydomonas reinhardtii Algae Gravitaxis Assay HTS Activity scoring rules:
The 96-well format cilia inhibitor HTS run at the PMLSC utilized % inhibition calculated from maximum (n=8) and minimum (n=8) plate controls, with a hit criteria of >/= 50% inhibition to identify active compounds.
Max control: Wild type cc124 cells in 1 % DMSO
Min control: mutant bld1 cells in 1 % DMSO
Cilia Inhibitor scoring rules:
PUBCHEM_ACTIVITY_OUTCOME
1 - Substance is considered inactive when the % inhibition is < 50 %
2 - Substance is considered active when % inhibition is >/= 50 %
3 - Substance activity outcome is inconclusive
PUBCHEM_ACTIVITY_SCORE
0-40 scoring range is reserved for primary HTS data
a) if the % inhibition is >/= 50 %, the score is 40.
b) if the % inhibition is < 50 %, the score is 0.

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: DDV248

Protocol: Four microliters of culture medium (RPMI 1640 with 0.5% w/v Albumax (GIBCO), 24 mM sodium bicarbonate and 10 ug/mL gentamycin) were dispensed by a Multi-drop Combi into white solid 1536-well plates (Grenier) and 23 nL compound was added by a pin tool. Four microliters of infected erythrocytes (2% hematocrit, 0.1% parasitemia final concentration) in culture medium were dispensed and the plates incubated for 48 hours at 37 C in 5% CO2. Two microliters of luciferase detection reagent was added and luminescence was detected by a ViewLux (PerkinElmer) reader

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: 20130620FPNS3INTERFERELOPAC

Protocol: Assay Overview:

The purpose of this biochemical assay is to determine the ability of compounds in Sigma-Aldrich's Library of Pharmacologically Active Compounds (LOPAC1280) to enhance or quench the fluorescence intensity of a Cy5-labeled substrate oligonucleotide bound to 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 of 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 Sigma-Aldrich's LOPAC-1280 set.

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: 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: FUM001

Protocol: Assay Protocol Summary:

For primary high-throughput screen, 3 ul of FH solution (containing 13.33nM human fumarate hydratase, 13.33IU/ml malic dehydrogenase, 0.2mM NAD, 0.067mg/ml diaphorase and 0.067mM resazurin in the assay buffer (50 mM Tris pH 8.0, 5 mM MgCl2, 0.01% Brij 3) was dispensed into each well in 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 20 nL of DMSO-solubilized compound (Cherrypick plates) to each 1536-well assay plate. Each compound was screened at five concentrations: 15 nM, 151 nM, 1.5 uM, 15.4 uM, 76.9 uM. Following compound transfer, plates were incubated in room temperature for 10 minutes. 1 uL of substrate solution containing fumaric acid (160 uM) was dispensed via BioRAPTR FRD to initiate the reaction. Plates were immediately transferred to a ViewLux microplate imager (PerkinElmer, Waltham, MA), and any resulting resorufin fluorescence was measured (exitation/emission, 525/598 nm) at 0 and 15 minutes. The exposure time is 1 second. Fluorescence from each well was normalized using enzyme-free and DMSO-treated control wells on each plate, and changes in fluorescence (delta RFU) were calculated using the difference in fluorescent signal for each well at 15 minutes versus 0 minutes.

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 activators are ranked higher than compounds that showed apparent inhibition.
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.