External ID: ULK1_INH_LUMI_1536_1X%INH PRUN

Protocol: Assay Overview:

The purpose of this biochemical assay is to identify compounds that act as inhibitors of Unc-51 Like Autophagy Activating Kinase 1 (ULK1). In this assay, ULK1 enzyme is incubated with test compounds and Atg13 and ATP to start the reaction. ULK1 specifically phosphorylates the Atg13 avidin tagged fusion protein. Phosphoryated Atg13 is then targeted by the anti-phospho Atg13 Ab. Rabbit IgG acceptor beads then recognize this complex. Streptavidin coated donor beads recognize the avidin tag from the Atg13 fusion protein. As designed, test compounds that act as ULK1 inhibitors in the presence of ATP will prevent the phosphorylation of Atg13, thereby leading to decreased luminescence signal from the beads. Compounds are tested in singlicate at a nominal test concentration of 5.96 micromolar.

Protocol Summary:
Prior to the start of the assay, 2 microliters of assay buffer (50mM Tris-Cl pH7.5, 20mM Magnesium chloride, 1mM DTT, 0.01% Triton X-100, 0.01% DMSO and 0.1mg/mL BSA) were dispensed into columns 1 thru column 3 of 1536 microtiter plates. Next, 2 microliters of assay buffer containing 2.5nM of ULK1 enzyme were dispensed into columns 4 thru column 48. Then, 30 nL of test compound in DMSO, Staurosporine (33uM final highest concentration) in DMSO, or DMSO alone (0.6% final concentration) were added to the appropriate wells and incubated for 15 minutes at 25 degrees Celsius. The assay was started by dispensing 1 microliter of assay buffer containing 100nM of Atg13 and 100uM of ATP to all wells. Plates were centrifuged and incubated for 120 minutes at 25 degrees Celsius. Next, 1 microliter of anti-pAtg13 (Final 0.5nM), EDTA (Final 20mM) and Anti-Rabbit IgG Acceptor beads (Final 16ug/mL) in 1X AlphaLISA detection buffer were dispensed into all wells. Plates were centrifuged and incubated for 60 minutes at 25 degrees Celsius. Then, 1 microliter of Streptavidin Donor Beads (Final 32ug/mL) was dispensed into all wells. Plates were centrifuged and incubated for 60 minutes at 25 degrees Celsius. Alphascreen signal was determined using an EnVision microplate reader ((PerkinElmer, Turku, Finland) at 680nm excitation and 570nm emission.




The percent inhibition for each compound was calculated as follows:

100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

Test_Compound is defined as wells containing ULK1 enzyme +Atg13/ATP in the presence of test compound
High_Control is defined as wells containing assay buffer (no enzyme) + Atg13/ATP and DMSO.
Low_Control is defined as the median of the wells containing test compounds.


PubChem Activity Outcome and Score:

Interval Cutoff

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-1, for inactive 1-0.

List of Reagents:

ULK1 enzyme (Sigma, part SRP0252-supplied by Derek Duckett)
Biotinylated-Atg13 (supplied by Derek Duckett)
Staurosporine (Enzo, part ALX-380-014)
Tris (Amresco, part 0497)
Magnesium chloride (Sigma, part M0631)
ATP (Sigma, part A6419)
DTT (Fisher, part BP172)
Triton X-100 (Sigma, part T9284)
DMSO (Fisher, part D159)
BSA (Calbiochem, part 126609)
EDTA (Fisher, part BP2482)
Anti-Atg13 pS318 Antibody (Rockland, part 600-401-C49)
AlphaScreen Rabbit IgG Detection Kit (Perkin Elmer, part 6760607M)
1536-well plates (Corning, part 7254)

Comment: Due to the size of the Scripps Molecular Screening Center compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the Scripps Molecular Screening Center.

External ID: SBCCG-A1016-RevErbaLBD-Primary-Assay

Protocol: This assay is to identify modulators of Rev-erb alpha protein binding to DNA

A. Materials:
REV-alpha_beta purified protein = Rastinejad lab
FITC-DNA = Rastinejad lab
Tris = Biorad (cat #161-0719)
DTT = Akron Biotechnology (cat #AK2948-0005)
5M NaCl = Sigma-Aldrich (cat #56546-1L)
Glycerol 99.5% = Acros (cat #327255000)
Tween 20 = Sigma-Aldrich (cat #P1379)
Corning high base black plates = Corning (cat #3724)
Molecular grade water = Cellgro (cat #46-000-CM)

B. Plate Map:
Negative (low) control in columns 1 and 2 is 6nM DNA and 35nM Protein, DMSO
Positive (high) control in columns 3 and 4, Protein at 750nM and 6nM DNA, DMSO
Test compound in columns 5 - 48, Protein at 35nM + 6nM DNA + test compound

C. Procedures:
Step#Description
1#Prepare 2X Rev-erb alpha protein stock and 2X DNA stock
2#Using LabCyte Echo, transfer xnL from a 2 mM Echo qualified plate containing test compounds into assay plate Col. 5 - 48. Add same volume of DMSO in col 1-4.
3#Spin plates at 1000 rpm for 1 minute in centrifuge.
4#Using the bioraptr, add 3 uL/well of (35nM protein control) to columns 1 and 2 and test compound wells.
5#Using the bioraptr, add 3 uL/well of Mix 2 (750nM protein) to col. 3-4 for the positive control
6#Using the bioraptr, add 3uL/well of Mix 3 (DNA) to col. 1-48.
7#Spin plates at 1000 rpm for 1 minute in centrifuge.
8#Incubate plates in the dark at room temperature for 90 minutes.
9#Set up Perkin Elmer EnVision as described in section Instrument setting.
10#Read plates on EnVision using FP Dual enhanced mirror, FP 480 excitation filter, FP-P-pol 535 and FP-S-pol 535 emissin filters

Comment: Actives were selected based on, % response = 45% or greater

External ID: CHEMBL4513082

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL4303835
ChEMBL Target Name: SARS-CoV-2
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: SARS-CoV-2 Screening Data

External ID: cmt-p4-fluc-fda_regid

Protocol: S16 Schwann cell PMP22 intronic element firefly luciferase assay
The FLuc-expressing S16 cells were seeded in white solid 1536-well plates at between 500-750 cells/4uL in DMEM medium containing 10% FBS, w/o phenol red. After overnight incubation at 37 degree Celsius/5% C02, 23 nL of compounds or DMSO were delivered to each well using a pin tool, followed by 24h-incubation at 37 degree Celsius/5% C02. Then 4 uL ONE-Glo luminescent substrate mix (Promega) was added to each well. Luminescence was measured on a ViewLux plate reader (Perkin Elmer). The % activity was determined by normalizing to the average readings of PTC124 (a positive control) - treated cells relative to DMSO-treated cells (0% Activity). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity.

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: PolK100

Protocol: Three microliters of reagents (buffer in column 3 and 4 as negative control and 10 nM Pol kappa in columns 1, 2, and 5-48) were dispensed into a 1536-well black solid-bottom 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 uL substrate (50 nM final concentration) were then 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: CHEMBL4303805

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL4303835
ChEMBL Target Name: SARS-CoV-2
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: SARS-CoV-2 Screening Data

External ID: CHEMBL4495582

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL4523582
ChEMBL Target Name: Replicase polyprotein 1ab
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

Data Source: SARS-CoV-2 Screening Data

External ID: HIV Cellular Data

Protocol: N/A

Comment: N/A

External ID: CHEMBL4303810

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL4303835
ChEMBL Target Name: SARS-CoV-2
ChEMBL Target Type: ORGANISM - Target is a complete organism
Relationship Type: N - Non-molecular target assigned
Confidence: Target assigned is non-molecular

Data Source: SARS-CoV-2 Screening Data

External ID: CHEMBL4808149

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL1865
ChEMBL Target Name: Histone deacetylase 6
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

Data Source: Fraunhofer Institute HDAC6 screening

External ID: CHEMBL4808150

Protocol: N/A

Comment: Target ChEMBL ID: CHEMBL1865
ChEMBL Target Name: Histone deacetylase 6
ChEMBL Target Type: SINGLE PROTEIN - Target is a single protein chain
Relationship Type: D - Direct protein target assigned
Confidence: Direct single protein target assigned

Data Source: Fraunhofer Institute HDAC6 screening

External ID: cmt-p4-fda-celltiter_regid

Protocol: Native S16 cells were seeded in white solid-bottom 1536 well plates at between 500-750 cells/6uL in DMEM medium containing 10% FBS, w/o phenol red. After overnight incubation at 37 degree Celsius/5% C02, 23 nL of compounds or DMSO were delivered to each well using a pin tool, followed by 24h-incubation at 37 degree Celsius/5% C02. Then one volume of CellTiter-Glo assay reagent (Promega) was added to each well for cell viability measured by a ViewLux plate reader (PerkinElmer). The % activity was determined by normalizing to the average readings from wells containing no cells relative to DMSO-treated cells (0% Activity). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity.

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, toxic compounds are ranked higher than non-toxic compounds.
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. Toxic 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: adst-fluc-km-fda-o1_regid

Protocol: Three microliters/well of substrate- buffer solution (0.01 mM D-Luciferin, 0.01 mM ATP, 50 mM Tris Acetate, 10 mM Mg Acetate, 0.01% Tween-20 and 0.038% BSA final concentrations) were dispensed into white solid-bottom 1536 well plates. Compounds were transferred to the plates in 23nl, and one microliter/well luciferase enzyme-buffer solution (10 nM P. pyralis luciferase and 50 mM Tris Acetate final concentrations) was dispensed yielding a final reaction volume of 4 ul/well. Luciferase luminescence was measured by a ViewLux plate reader within 5 minutes of enzyme-buffer addition. The % activity was determined by normalizing to the average readings of PTC124 (a positive control) relative to DMSO (0% Activity). The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity.

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.