Fluorescence polarization acridine orange assay to identify DNA binding small molcules Measured in Biochemical System Using Plate Reader - 7014-02_Inhibitor_Dose_CherryPick_Activity
Assay Overview: This screening project aims to identify specific inhibitors of the EWS/Fli activity, a fusion protein involved in Ewing sarcoma. To eliminate DNA binding as an non-desired mechanism of action, the ability of compounds to bind DNA is tested in this assay. Compounds are pinned into reaction mix containing acridine orange and salmon sperm DNA in 384-well plates, for 20 minutes before measuring the fluorescence polorization of acridine orange at excitation/emission 480/535 nm (S/P ratio). Compounds binding DNA are decreasing the excitation/emission 480/535 S/P polarization ratio. ..more
BioActive Compounds: 14
Keywords: cancer, counter screen, acridine orange, DNA binding, EWS/Fli
Assay Overview: This screening project aims to identify specific inhibitors of the EWS/Fli activity, a fusion protein involved in Ewing sarcoma. To eliminate DNA binding as an non-desired mechanism of action, the ability of compounds to bind DNA is tested in this assay. Compounds are pinned into reaction mix containing acridine orange and salmon sperm DNA in 384-well plates, for 20 minutes before measuring the fluorescence polorization of acridine orange at excitation/emission 480/535 nm (S/P ratio). Compounds binding DNA are decreasing the excitation/emission 480/535 S/P polarization ratio.
Expected Outcome: Compounds decresing the fluorescent polarization S/P ratio at a concentration below 20uM will be considered hits and should be discarded for further analysis.
Dispense 30 uL reaction mix and positive control with combi and combi nL into specific wells on 384-well plates based on plate design.
Pin 100 nL/well of compounds.
Dispense 1.5 nL postive control (Mitoxantrone, Axxora, ALX-400-050-M050) with combi nL to get 10 uM final conc.
Incubate at room temperature for 20 minutes.
Read on Envision at 480/535 S and P nm with D505fp/D535 dichroic mirror.
Reaction mix: 6 ug/mL Salmon sperm DNA (Invitrogen, 15632-011) and 50nM acridine orange (Invitrogen, A1301) in HEN buffer (10 mM HEPES, pH 7.5; 1 mM EDTA pH 7.5; 100 mM NaCl)
Positive control: 10 uM of Mitoxantrone in reaction mix
PRESENCE OF CONTROLS: Neutral control wells (NC; n=18) and positive control wells (PC; n=18) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
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 (v10.0.2):
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.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold -35.0
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.
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
Assay Format: Biochemical
Assay Format: Biochemical
Assay Type: Binding
* Activity Concentration. ** Test Concentration.
Data Table (Concise)