RAD52: DNA binders Measured in Biochemical System Using Plate Reader - 7018-02_Inhibitor_Dose_CherryPick_Activity
The goal of this project is to develop specific small-molecule inhibitors of human RAD52, one of the key homologous recombination (HR) proteins. Previously we ran a primary screen and identified putative RAD52 inhibitors. To rule out DNA binding as an undesired mechanism of action, we tested the ability of these compounds to bind DNA in this assay. Basically, compounds in varied doses were more ..
BioActive Compounds: 85
Homologous Recombination, DNA repair, RAD52
The goal of this project is to develop specific small-molecule inhibitors of human RAD52, one of the key homologous recombination (HR) proteins. Previously we ran a primary screen and identified putative RAD52 inhibitors. To rule out DNA binding as an undesired mechanism of action, we tested the ability of these compounds to bind DNA in this assay. Basically, compounds in varied doses were pinned into reaction mix of acridine orange and DNA on 384-well plates, and the reactions were incubated at room temperature for 20 minutes before fluorescence polorization was read on EnVision (Perkin Elmer) at ex/em 480/535 S/P nm.
DNA binding of test compounds will result in loss of signal.
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) 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 and 50nM acridine orange 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=32) and positive control wells (PC; n=32) 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 (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): 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)