ARNT-TAC3: AlphaScreen HTS to detect disruption of ARNT/TAC3 interactions Measured in Biochemical System Using Plate Reader - 2158-01_Inhibitor_SinglePoint_HTS_Activity
Keywords: Aryl hydrocarbon receptor nuclear translocator (ARNT), coil coil coactivators (CCC), basic helix-loop-helix/per-ARNT-Sim (bHLH/PAS), hypoxia, xenobiotic exposure ..more
BioActive Compounds: 2589
Depositor Specified Assays
Keywords: Aryl hydrocarbon receptor nuclear translocator (ARNT), coil coil coactivators (CCC), basic helix-loop-helix/per-ARNT-Sim (bHLH/PAS), hypoxia, xenobiotic exposure
Assay Overview: The Alphascreen protein/protein interaction (Perkin Elmer) is used for the primary assay. This in vitro assay relies on luminescence proximity between "donor" and "acceptor" beads to report on complex formation, requiring that two beads localize within 200 nm for the singlet O2-mediated process to occur in high efficiency. His6-ARNT PAS-B (~110 amino acids) and a GST- tagged fragment of the TACC3 coactivator (~40 residues) are added together along with compound. Using affinity tags to recruit beads into close proximity, compound mediated decreases in luminescence are monitored to indicate candidates that may be disrupting the complex. Optimal protein concentrations rely on a 2:1 ratio of ARNT PAS-B to TACC3 dimer (=1:1 ratio of ARNT:TACC3 chain). The positive control is the His-ARNT protein alone.
Expected Outcome: Compounds that interfere with ARNT PAS-B binding with TACC3 coactivator will show a decrease in luminescence.
MLPCN ARNT/PASB Protocol
1.His-ARNT PAS-B (In Alpha storage buffer, 368 microM)
2.GST-TACC3 E629A dimer (In Alpha storage, 113 microM)
3.Ni2+ acceptor beads, 5 mg/ml (PerkinElmer, Nickel Chelate AlphaLISA(R) Acceptor Beads 5 mg, Product number: AL108M)
4.GST donor beads, 5 mg/ml (PerkinElmer, AlphaScreen(R) Glutathione Donor Beads, Product number: Product number: 6765302 for 25mg pack)
5.Alpha storage buffer: 50 mM Tris, pH 7.5; 100 mM NaCl; 10% glycerol; 1 mM DTT, 0.02 % Tween 20
6.Alpha screening buffer: 50 mM Tris, pH 7.5; 100 mM NaCl; 1% glycerol; 0.02 % Tween 20, 1 mM DTT (freshly added); 0.1% BSA (freshly added)
7.Assay ready Plates (ARPs): Aurora 1536K High Base White Plate (solid bottom, square well, nonsterlie, untreated) with compounds added to each well prior to screen using Labcyte Echo acoustic transfer.Stored at -20 degrees Celsius prior to use.
1.Bring ARPs to room temperature. Store ARPs in Liconic incubator (set at room temperature) with lids
2.Add 2.25 microL His-ARNT (1.336 microM) to each well of full 1536 plate using Beckman Coulter BioRaptr* (final concentration 0.668 microM)
3.Add 2.25 microL GST-TACC3 E629A dimer (0.668 microM) to neutral control wells and assay wells using Beckman Coulter BioRaptr* (final concentration 0.334 microM)
4.Add 2.25 microL of Alpha screening buffer to positive control wells using BioRaptr*
5.Incubate for 1 hr. at room temperature in Liconic
6.Add 3 uL/well of Alphabead mixture (12.5 microg/mL) using CombiNL (final concentration 5 microg/mL)
7.Incubate for 1 hr. at room temperature in Liconic
8.Read plate using on Envision plate reader using protocol: ARNT Alphascreen 1536
9.Take plate to trash
*BioRaptr used with cooled jackets around storage bottles to keep reagents cooled.
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 50.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 50.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE <= AT, and PCT_ACTIVE_REP >= PAR_T
** Test Concentration.
Data Table (Concise)