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: 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: HMS791

Protocol: Prior to screening, FITC LANA1-23 was stored lyophilized at -80 degC and freshly purified chicken nucleosomes were stored at 4 degC in 20 mM Tris pH 7.5, 600 mM NaCl, 0.2 mM EDTA, 0.5 mM B-Mercaptoethanol.

On the day of screening, FITC LANA1-23 was resuspended at 50 uM in TEN-BT buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 2.5 mM NaCl, 5 mM Beta-mercaptoethanol, 0.01% Triton-X-100) and diluted to a final concentration of 50 nM in TEN-BT buffer plus 240 nM of purified nucleosomes (480 nM LANA peptide binding sites). 30 uL per well were dispensed in column 1-22 in Corning #3575 black 384 well plates.

Wells in column 23 contained 30 uL of the same mixture (for pilot screen) or with the addition of 10 uM monensin (for HTS) as a negative control. In column 24, 1250 nM unlabeled WT LANA1-23 peptide (for pilot screen) or 10 uM mitoxantrone (for HTS) was added to the wells as a positive control. Compounds were transferred into wells via stainless steel pin array (100 nL) and the reaction was incubated at room temperature for 10 to 45 minutes (stable for up to 2 hours). Library plates were screened in duplicate, with both assay plates in a given set prepared on the same day.

Following a room temperature incubation of 10 to 45 minutes, the assay is read on a EnVision plate reader using a 480 nM excitation filter, 535 nM S and P emission filters and D505fp/D535 dichoric mirror. mP value for FP measurement = 1000*(S-G*P)/(S+G*P) where S= , P=, G= G-factor. The G Factor = 1.

Comment: LANA 1-23 peptide containing the first 23 amino acids of the LANA protein from Kaposi's sarcoma herpesvirus (KSHV) was synthesized with an N-terminal FITC via a beta alanine linker and HPLC purified (peptide sequence: [FITC]-Beta alanine-MAPPGMRLRSGRSTGAPLTRGSC-[NH2]).

Data analysis: Z-scores were calculated for each replicate well using the mean and standard deviation of plate experimental well FP values. Compounds were considered active if the Z-score for both replicates < -2. Wells with high total fluorescence intensity (high S and P channel values) were excluded from further consideration. Activity scores were calculated based on replicate average FP Z-scores. For wells with average Z-score >= 0, the activity score was set to 0. For wells with replicate average Z-score < 0, replicate Z-score was divided by 4 and multiplied by -100. The replicate average was then used to determine the well activity score. Values > 100 (replicate average Z-score < -4) were set to 100.

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: HMS979

Protocol: Cation-adjusted Mueller Hinton II broth supplemented with 0.005% Tween-80 was inoculated with a single colony of S. aureus RN4220. Following overnight incubation, the culture was diluted 1:100 into fresh medium, and incubation was continued until the bacterial suspension reached an optical density at 600 nm (OD600) of 0.6, corresponding to a bacterial density of 5 x 10E8 colony forming units (CFU)/mL. An aliquot of this culture was diluted 1:400 with fresh medium and stored on ice until use. Assay plates were prepared for compound transfer in quadruplicate to enable screening of compounds at two temperatures in duplicate. The plate layout was as follows: wells in columns 1-22 were loaded with culture medium at 25 microL/well; wells in column 23 contained the screening negative control (medium only); wells in column 24 contained the screening positive control (medium containing 25 microg/mL chloramphenicol). 300 nL of experimental compounds were transferred by stainless steel pin array from library plate to assay plates.

25 microL of the bacterial suspension was added to each assay well. Assay plates were incubated at 42 degrees C in humidified chambers (humidity > 85%). After 20 h, the OD600 was measured using a plate reader in absorbance mode.

Comment: Data analysis description:

Absorbance values at 42 degrees C and 30 degrees C for each replicate were normalized to positive and negative control plate average absorbance, and replicate normalized values were averaged to calculate an average relative absorbance at both temperatures. A substance was considered a temperature-dependent positive at 42 degrees C if average relative absorbance <= 50 and the difference between relative absorbance at 30 degrees C and 42 degrees C >= 20 (relative absorbance 30 degrees C - relative absorbance 42 degrees C >= 20). A substance was considered a temperature-independent positive if relative absorbance at both 30 degrees C and 42 degrees C < 10.

Activity scores were calculated using average relative absorbance at both temperatures. Average relative absorbance <= 0 was scored as 100 for activity; average relative absorbance >= 100 was scored as 0 for activityy. Average relative absorbance between 0 and 100 was subtracted from 100 to generate activity scores for intermediate values (i.e. average relative absorbance = 40 corresponds to an activity score of 60).

Note that since this is treated as a panel assay, the query for active compounds includes many that were considered active at both temperatures. The final Pubchem activity score is the activity score at 42 degrees C, and compounds scored as active (PUBCHEM_ACTIVITY_OUTCOME = 2) include both temperature-dependent and temperature-independent active substances.

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.

External ID: HMS750

Protocol: The stalled elongation complex used as a substrate in the screening assay was generated by pre-incubating 600 nM 3Dpol with 12 nM 5' fluorescein labeled 8-6 PETE RNA and 240 nM ATP for 30 minutes at room temperature in 50 mM HEPES, pH 7.0, 40 mM NaCl, 1.5 mM magnesium acetate, 60 microM ZnCl2, 4 mM DTT, and 0.1% Igepal detergent. This solution was dispensed into 384-well microplates (Corning 3710) at a volume of 25 microL/well. Experimental compounds were added by robotic pin transfer at 100 nL/well.

After an incubation time of ~60 min, the total fluorescence and fluorescence polarization signals from the labeled RNA were measured with Perkin Elmer EnVision microplate readers. This first reading provided data indicating whether compounds interfered with RNA binding to the stalled elongation complex. Polymerase elongation activity was then tested by adding 5 microL of a solution containing GTP, CTP, and UTP at 1.2 microM each, resulting in a final concentration of 200 nM for each of the four NTPs and 16.7 microg/ml for the compounds. This allowed for elongation to the end of the RNA template, and assay data were obtained with a second reading done after an incubation period of ~60 min, which is four times longer than the ~15 min needed for elongation under non-inhibited conditions.

Comment: Potential inhibitors were identified using a correlation plot combining standard Z-scores with an elongation efficiency measurement. A Z-score for each compound was calculated by the normal method of Z=(FPcompound-FPplate_mean)/StdDevplate_mean. An elongation efficiency value (FP %Elongation) was then calculated as E=(FPcompound-FPpositive)/(FPnegative-FPpositive), which reflects how the FP signal obtained in the presence of a compound compared to those of the unelongated positive controls (FPpositive) and fully elongated negative controls (FPnegative). Compounds that gave a FP signal greater than the starting material but less than the fully elongated controls thus had E values between 0% and 100%, while compounds that caused the RNA to dissociate from 3Dpol had negative E values due to the FP value associated with free RNA being lower than the unelongated FPpositive value. Finally, the elongation reaction also results in an increase in the total fluorescence intensity due to deprotonation of the fluorescein as it approaches the positively charged polymerase surface, providing an additional assessment of elongation. Compounds that resulted in total fluorescence %Elongation higher than that of the fully elongated control samples were rejected from the analysis. Positives were defined as having experimental FP Z-scores < -0.5 and FP %Elongation < 90%. Activity scores were calculated based on FP %Elongation. FP %Elongation <= 0 was scored as 100 for activity; FP %Elongation >= 100 was scored as 0 for activity. FP %Elongation values between 0 and 100 were subtracted from 100 to generate activity scores.