External ID: MH081226: Primary HTS for Inhibitors of a Novel Necrotic Cell Death Pathway # Jurkat FADD-/- Model.

Protocol: Necroptosis Assay Protocol TNF-alpha Induced Cell Death in Jurkat FADD-/- Cells.

Based on the assay development experiments performed by the PMLSC the following assay conditions were selected for the 384-well TNF-alpha Jurkat FADD-/- necroptosis screen:
1. 20,000 Jurkat FADD-/- cells/well were seeded in 40 uL of complete media in white opaque 384-well microtiter plates.
2. Plate controls, compounds (10 uM final in well) and 40 ng/mL TNF-alpha were added in a single addition and cells were incubated for an additional 24 hrs at 37#C and 5% CO2.
3. 25 uL of Cell Titer Glo reagent was added to each well and the resulting luminescence measurement of cellular ATP levels was read on the Envision after 15 minutes at ambient temperature.

Materials:
1. Complete Media: RPMI1640 with L-glutamine, Penicillin-Streptomycin, 10% Newborn calf serum
2. Jurkat FADD-/- Cells: 20,000 cells/well in 40 μL complete media
3. TNF-alpha: 50 ug/mL stock in DMSO; final concentration = 40 ng/mL
4. Cell Titer Glo: use at 25 μL/well

Protocol:
1. Jurkat FADD-/- cells are seeded at 20,000 cells/well in 40 uL of complete media into 384-well white opaque-bottom polypropylene plates (Greiner Bio-one Cat #781080).
2.Compounds (10 uM final concentration) are added to the respective wells in a total volume of 10 uL/well.
3. Maximum and minimum controls (DMSO at 0.18% final concentration and TNF-alpha at 40 ng/mL final concentration respectively) are added to assay plates at 20 uL/well
4. TNF-alpha (40 ng/mL final concentration) is added at 10 uL/well to respective wells with compounds.
5. Cell plates are incubated overnight at 37#C and 5% CO2.
6. 25 uL Cell Titer Glo is added to each well and the luminescence signal is read on the Envision after 15 minutes.

Plate Controls:
Max: 0.18% DMSO in complete media
Min: 40 ng/mL TNF-alpha0.08% DMSO) + 0.1% DMSO in complete media (final DMSO concentration = 0.18%)

Comment: The TNF-alpha induced Jurkat FADD-/- necroptosis Assay HTS run at the PMLSC utilized % activation calculated from maximum (n=32) and minimum (n=24) plate controls, with a hit criteria of >/= 50% activation to identify active compounds.

TNF-alpha Jurkat FADD-/- necroptosis Assay Activity scoring rules:

PUBCHEM_ACTIVITY_OUTCOME

1 - Substance is considered inactive when the % activation is < 50%
2 - Substance is considered active when % activation is >/= 50%
3 - Substance activity outcome is inconclusive

PUBCHEM_ACTIVITY_SCORE

0-40 scoring range is reserved for primary HTS data
a) if the % activation is >/= 50%, the score is 40.
b) if the % activation is < 50 %, the score is 0.

Definition of a Hit:
Rapid HTS screen ≥ 50% inhibition of death at 10 uM.
Confirmation of ≥ 50% inhibition of TNF-alpha induced Jurkat FADD-/- cell death in 2 independent tests.
Concentration Response AC50 < 20 uM in the Jurkat FADD-/- cell death model.
Structural Verification
Indications of Specificity & Selectivity ~ PubChem X-target Query
Evidence of SAR.

External ID: MH083223 Targeting HIV-1 Nef with Small Molecules

Protocol: HIV Hck:Nef HTS Protocol
1. Recombinant Hck-YEEI protein, was purified to homogeneity from Sf-9 insect cells
2. Recombinant HIV-1 Nef (SF2 strain), was purified to homogeneity from E. coli
3. Z-Lyte assay reagents were purchased from (Invitrogen).

Several Hck and Nef protein preparations provided by the assay provider were utilized in the HTS campaign, and several Hck:Nef protein ratios were utilized 10 ng + 50 ng, 12.5 ng + 75 ng, 15 ng + 75 ng, and 20 ng + 100 ng in the 384-well HTS assay.

HTS Protocol:
1. Thaw compound plates, 2uL of 1 mM in 100% DMSO.
2. Spin compound plates down, 5 min 50 x g.
3. Dilute 2 uL 1mM compounds with 23ul of water on Flex Drop (80 uM, 8% DMSO).
4. Mix and Transfer 2.5ul of compound to assay plate on EP3.
5. Transfer 5ul of Max (2.5ul DMSO and 2.5ul Hck:Nef) and Min (2.5ul DMSO and 2.5ul Hck/1x kinase) controls (from pre-made control plates) to assay plate on EP3.
6. Add 2.5ul of Hck:Nef to assay plate(340 wells), spin down and incubate for 30 min at ambient temperature (40 uM, 4% DMSO).
7. Add 5 uL of ATP/Tyr complex to whole plate on MicroFlow, spin down plates and put on shaker to incubate at ambient temperature for 50 min (20 uM, 2% DMSO).
8. Add Development buffer to whole plate on MicroFlow, spin down plates and put on shaker to incubate at ambient temperature for 60 min.
9. Add Stop reagent to whole plate on MicroFlow, spin down.
10. Read Donor to Acceptor FRET Emission Ratio on M5 plate reader within 2 hrs. Excitation of the donor fluorophore at 400 nm, Donor to Acceptor FRET Ratio = ratio of Coumarin emission 445 nm to Fluorescein emission 520 nm.

Comment: Hck-Nef Assay HTS Activity scoring rules:
The 384-well format Hck-Nef inhibitor HTS run at the PMLSC utilized % inhibition calculated from maximum (n=32) and minimum (n=24) plate controls, with a hit criteria of >/= 50% inhibition to identify active compounds.
Max control: 2.5ul DMSO and 2.5ul Hck:Nef
Min control: 2.5ul DMSO and 2.5ul Hck/1x kinase (No Nef)
Hck-Nef 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: 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: MH080376 Biochemical HTS for Inhibitors of the Proprotein Convertase Furin.

Protocol: Biochemical Furin HTS Assay protocol

Reaction Buffer Concentrations (final concentrations): 50 mM Hepes pH 7.5, 1 mM CaCl2, 1 mM beta-mercaptoethanol , 0.2 mg/ml BSA.

Substrate Stock Solution: 10 mM pERTKR-AMC in DMSO; stored in aliquots at -20 oC.

rhFurin714 prep (5.2 units/uL)Stored in aliquots at -80oC.

Furin Assay Protocol

The assay involves three liquid transfer steps of 5 uL each of 30 uM compound in 1% DMSO (10 uM final), 1 unit/5 uL rhFurin714 in 3X reaction buffer and 5 #L of 30 uM pERTKR-AMC substrate (10 uM final).

The assay plates used are Greiner 384-well, flat-bottom, low volume, black polystyrene plates (VWR catalog # 784076).

1. Add 5 uL of compound/controls to each well.
2. Add 5 uL of rhFurin714 in 3X buffer (1.0 units/well final).
3. Add 5 uL of 30 uM pERTKR-AMC fluorigenic substrate (10 uM/well final).
4. Incubate the plates for 1 hr at room temperature.
5. Stop the reaction by adding 5 uL of 1M Acetic acid.
6. Measure the amount of fluorigenic AMC released on the SpectraMax M5 using Ex = 345nm; Em = 440nm; cut-off 420 nm.

Comment: Active Criteria, Secondary Assay Plan, Hit and Lead Criteria.

Furin HTS Activity scoring rules:

The Furin inhibitor HTS run at the PMLSC utilized % inhibition calculated from maximum (n=32) and minimum (n=24) plate controls, with a hit criteria of >/= 25% inhibition to identify active compounds.

Furin Inhibitor scoring rules:

PUBCHEM_ACTIVITY_OUTCOME

1 - Substance is considered inactive when the % inhibition is < 25 %
2 - Substance is considered active when % activation is >/= 25 %
3 - Substance activity outcome is inconclusive

PUBCHEM_ACTIVITY_SCORE

0-40 scoring range is reserved for primary HTS data
a) if the % inhibition is >/= 25 %, the score is 40.
b) if the % inhibition is < 25 %, the score is 0.

Definition of Hit Criteria:
It is anticipated that all Furin inhibitor HTS actives will be confirmed in duplicate at the primary HTS concentration of 30 uM.
Furin inhibitor actives confirmed in the primary HTS format will then be run in 10-pt IC50 concentration response curves.
Confirmed hits will be subjected to structural confirmation by LCMS.

Secondary assay testing paradigm: Confirmed concentration dependent Furin inhibitors will be tested in the HeLa pcFur1.6 cell based ELISA assay.

External ID: MH083154

Protocol: Materials
MATERIALS
MEF-GFP-LC3 cells (provided by Dr. Xiao-Ming Yin, University of Pittsburgh)
384 well flat bottom microplates, collagen-coated (Falcon Biocoat)
DMEM growth medium (Gibco), supplemented with 10% Fetal bovine serum (HyClone) and antibiotics
Thapsigargin (Molecular Probes T-7459), dissolved in DMSO at 1 mM and frozen in aliquots at -20oC.
DMSO (Sigma 15,493-8)
Formaldehyde, 37 wt. % solution in water, A.C.S. reagent (Sigma 252549)
Test compounds plated into 384 well microplates (Greiner polypropylene).
Hanks balanced salt solution (HBSS, Fisher SH3026802)
Hoechst 33342 (Molecular Probes/Invitrogen H-1399)

PROTOCOL
Day 1. Cell seeding.
Plate 5,000 MEF-GFP-LC3 cells/40 [micro]L complete growth medium into the wells of collagen-coated 384 well microplates and incubate overnight at 37oC, 5% CO2.
Day 2. Compound treatment.
Thaw compound plates containing 2 [micro]L of 1 mM drug in DMSO.
Reconstitute wells A3:P22 of library compound plates in 38 [micro]L complete growth medium. Intermediate concentrations are 50 [micro]M compound and 5% DMSO
Load appropriate control wells on reconstituted library compound plates with 40 [micro]L of positive and negative controls (5% DMSO) and 5 [micro]M thapsigargin in 5% DMSO (Biomek2000, Perkin Elmer Multiprobe).
Transfer 10 [micro]L of treatment solution from reconstituted library compound plates to assay plates. (Perkin Elmer Janus MDT) and incubate for 18h at 37oC, 5% CO2. The final assay concentration will be 10 [micro]M compound and 1% DMSO.

Day 3. Fixation, staining, and analysis.
Prepare fixative (10.7% formaldehyde and 26.7 [micro]g/ml Hoechst 33342 in HBSS).
Dispense 30 [micro]L of fixative directly into all plates (Titertek MAP-C2).
Incubate for 30 min at room temp. under dimmed light.
Aspirate off fixative and wash plates three times with 50 [micro]L PBS (MAP-C2).
Seal plates and analyze on the ArrayScan Vti (Thermo Fisher Cellomics) for autophagosomal GFP-LC3 accumulation.
Acquire images in two channels (DAPI/FITC) using a 10x objective and an XF100 filter set on the ArrayScan VTi.

ANALYSIS
Analyze images by the Compartmental Analysis Bioapplication, configured to detect Hoechst 33342 stained nuclei, cytosolic GFP expression, and autophagosomes as GFP-expressing punctate objects located in the cytosol. First, nuclear and cytosolic areas were separated to avoid nuclear punctate objects to be included. Punctate structures were then detected in the cytosol by thresholding over local background. A selection threshold was then set using DMSO-treated cells to calculate the percentage of cells positive for autophagosome formation.

Comment: HCS parameters reported
1. %HIGH_RingSpotCountCh2: The percentage of cells containing GFP spots (Channel 2). The percentage of responders is computed on a plate by plate basis.
2. Z-score_%HIGH_RingSpotCountCh2: Z-score of the percentage of cells containing GFP spots (Channel 2).
3. HCS_cmpd_conc: Primary HCS compound concentration in [micro]M
4. MEAN_RingAvgIntenCh2. The average GFP intensity in the cytosol (Channel 2). The average intensity is reported on a per cell basis.
5. MEAN_RingSpotTotalAreaCh2. The total (integrated) intensity of GFP spots per cell (Channel 2). The total spot intensity is reported on a per cell basis.
6. SelectedObjectCountPerValidField. The number of nuclei per imaging field.
7. % Toxicity. % toxicity = 1-(SelectedObjectCountPerValidField in sample well / average SelectedObjectCountPerValidField of all MIN controls on the plate)*100
8. Assay Date : Date the HTS assay was performed

The high-content cell-based screen for modulators of autophagy conducted by the PMLSC utilized a Z-score statistical scoring method to identify active compounds (Brideau et al., 2003)
Brideau, C., Gunter, B., Pikounis, B., and Liaw, A. (2003). Improved statistical methods for hit selection in high-throughput screening. Journal of Biomolecular Screening 8, 634-647. 14711389

Target activity score (Z-score_%HIGH_RingSpotCountCh2)
The Z-score for a compound was computed on a plate by plate basis. The Z-score for the percentage of cells containing autophagosomes in Channel 2 (Xi) was defined as Xi = (Xi - Xm)/Sm, where Xm is the mean of all the percentage of cells containing autophagosomes values in wells A3:P22 on the microplate, and Sm is the standard deviation of these values. A cut-off Z-score of greater than 3 was selected as the definition as the active criterion.

Fluorescent outliers. (MEAN_RingAvgIntenCh2)
The average GFP intensity per cell was calculated on a per cell basis. A cut-off score of 1500 was selected as the definition for a fluorescence outlier.

Cytotoxicity outliers (% toxicity)
% toxicity was computed on a plate by plate basis. % toxicity was defined as 1-(cell density in sample well / average cell density of all MIN controls on the plate)*100. A cut-off score of more than 80% was selected as the definition for a cytotoxic compound.


Definition of an active compound
Z-score_%HIGH_RingSpotCountCh2 > 3, MEAN_RingAvgIntenCh2 > 1500, and % toxicity < 80%

PUBCHEM_ACTIVITY_OUTCOME
1 inactive. Substance is considered inactive when the Z-score_%HIGH_RingSpotCountCh2 > 3 at 10 [micro]M.
2 active. Substance is considered inactive when the Z-score_%HIGH_RingSpotCountCh2 > 3, MEAN_RingAvgIntenCh2 < 1500, and % toxicity < 80% at 10 [micro]M

PUBCHEM_ACTIVITY_SCORE
0-40 range is reserved for primary HTS data
a)If the substance is considered active the score is 40
b)If the substance is inactive the score is 0

Secondary assay testing paradigm:
Confirmation of inhibition of autophagosome formation in 2 independent tests at 10 [micro]M.
Concentration Response IC50 < 10 [micro]M in the autophagy inducer HCS assay.
Structural Verification
Indications of Specificity & Selectivity ~ PubChem X-target Query
Evidence of SAR.

Confirmed concentration dependent actives would be tested for A) autophagosome formation in a different cell line stably expressing LC3-GFP and B) for long lived protein degradation.