AID	Name	Type	Comment
602352	Broad Institute Identification of Inhibitors of RAD54 Probe Project	summary

Keywords:
RAD54, Branch migration, Holliday Junction, homologous recombination, DNA repair, cancer

Assay Overview:
The goal of this proposal is to develop specific small-molecule inhibitors of human RAD54, one of the key homologous recombination (HR) proteins. Basically, RAD54 and DNA that forms Holiday Junctions are added according to plate design with BioRAPTR (Beckman) to respective wells of 1536-well assay ready plates (Aurora 19180) that contain 2.5 nL/well of 10 mM compound. The reaction is incubated at room temperature for 40 minutes. Fluorescence is then read on Viewlux (Perkin Elmer) at ex/em 480/540 nm.



Expected Outcome: Inhibitors of RAD54 will result in loss of signal.

1.#Reaction is started by Dispensing 0.6 uL/well of RAD54 Mix, 0.6 uL/well of DNA Mix, and 1.2 uL/well of Reaction Buffer in respective wells with BioRAPTR (Beckman) according to plate design in 1536-well assay ready plates (Aurora 19180) that contain 2.5 nL/well of 10 mM compound.
2.#Incubate at room temperature for 60 minutes.
3.#Read fluorescence at ex/em 480/540 nm on Viewlux


Reaction Buffer
Concentrations Tris base pH7.525mMMagnesium Acetate3mMBSA0.1mg/mlDTT2mMCreatine Phosphate15mMCreatine Phosphate kianse10Us/mLATP2mM
RAD54 Mix:
120 nM RAD54 (Provided by A Mazin, Drexel University College of Medicine) in Reaction Buffer

DNA Mix:
120 nM of the annealed Oligos (33/1337 and 265/1265) in Reaction Buffer. The oligos are as follows.

AVM#337-F, 60-mer: /56-FAM/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) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
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.

PUBCHEM_ACTIVITY_SCORE:
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 40.

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 0.5.

PUBCHEM_ACTIVITY_OUTCOME:
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 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

tSamples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

Grant Number: R03 DA033981-01A1