Identification of Rad54 Inhibitors - Ruling Out Flurescence Quenchers Measured in Biochemical System Using Plate Reader - 2159-03_Inhibitor_Dose_CherryPick_Activity
RAD54, branch migration, Holliday Junction, homologous recombination, DNA repair, cancer, counter screen ..more
RAD54, branch migration, Holliday Junction, homologous recombination, DNA repair, cancer, counter screen
The goal of this project is to develop specific small-molecule inhibitors of human RAD54, one of the key homologous recombination (HR) proteins. Previously we ran a primary screen and identified putative RAD54 inhibitors with DNA substrate labelded with fluorescein-amidite (FAM). To rule out false positives that quenched the fluorescein, we repeated the assay under the same conditions except that the DNA substrate was lebeled with Cy5. Basically, compounds in varied doses were pinned into reaction mix of Rad54 on 384-well plates, and the reactions were initiated with addition of DNA substrate. Reactions were incubated at room temperature for 60 minutes before fluorescence was read on EnVision (Perkin Elmer) at ex/em 480/540 nm.
Expected Outcome: Inhibitors of RAD54 will result in loss of signal.
RAD 54 Retest Protocol for 384 well plates:
1. Dispense 20 uL/well of RAD54 mix and positive control, the RAD54 mix without RAD54, with combi and combi nL respectively to specific wells according to plate design.
2. Pin 100 nL/well of 10 mM compound.
3. Add 1 uL/well of DNA mix with combi nL.
4. Incubate at room temperature for 60 minutes.
5. Read fluorescence at ex/em 480/540 nm on Envision
Concentrations Tris base pH7.525mMMagnesium Chloride3mMBSA0.1mg/mlDTT2mMCreatine Phosphate15mMCreatine Phosphate kianse10Us/mLATP2mM
30 nM RAD54 (Provided by A Mazin, Drexel University College of Medicine) in Reaction Buffer
628 nM of the annealed Oligos (337/1337 and 265/1265) in 25 mM TrisCl, pH7.5. The oligos are as follows.
AVM#337-C, 60-mer: /56-Cy5/CAC TGT GAT GCA CGA TGA TCG ACG ACA GTA GTC AGT GCT GGG TCA ACA TCT GTA TGC AGG
AVM #1337-BHQ: AGC ACT GAC TAC TGT CGT CGA TCA TCG TGC ATC ACA GTG/3BHQ_1/
AVM #1265, 60-mer: CAC TGT GAT GCA CGA TGA TTG ACG ACA GTA GTC AGT GCT TTT TTT TTT TTT TTT TTT TTT
AVM #265, 60-mer: CCT GCA TAC AGA TGT TGA CCC AGC ACT GAC TAC TGT CGT CAA TCA TCG TGC ATC ACA GTG
PRESENCE OF CONTROLS: Neutral control wells (NC; n=132) 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 (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.
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)