ARNT-TAC3: AlphaScreen HTS to detect disruption of ARNT/TAC3 interactions Measured in Biochemical System Using Plate Reader - 2158-01_Inhibitor_Dose_CherryPick_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: 967
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 buffer, 113 microM)
3.Ni2+ acceptor beads, 10 mg/ml (PerkinElmer, Nickel Chelate AlphaLISA(R) Acceptor Beads 5 mg, Product number: AL108M)
4.GST donor beads, 10 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 (25 microg/mL) using CombiNL (final concentration 10 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; n=36) and positive control wells (PC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
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.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)