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.