AID	Name	Type	Comment
1385	Homologous recombination - Rad 51	screening	Rad51 HTS (AID 1385)
1437	Homologous recombination_Rad 51_dose response_2	confirmatory
1436	Homologous Recombination_Rad51_DNA binding assay	confirmatory

Project Title: A screen for modulators of human Rad51, a key DNA repair protein

Application Number: MH084119

Assay Submitter: Dr. Alex Mazin

Submitter Institution: Drexel University

Screening Center Name: Penn Center for Molecular Discovery (PCMD)

Principal Investigator of Screening Center: Scott Diamond

Ionizing radiation (IR) and inter-strand cross-linking agents (ICL) induce DNA double-stranded breaks (DSB). DSB are the most harmful type of DNA damage, which cause genome instability, cancer, genetic diseases, and premature aging. The system of homologous recombination (HR) is responsible for repair of DSB repair in all organisms including humans. Therefore, HR acts primarily as a tumor suppressor. However, HR may also protect cancer cells against IR and ICL that are commonly used in anti-cancer therapy. In addition, HR is required for cell proliferation, the function of which is essential for tumorigenesis. Consequently, we propose to specifically inhibit HR during anti-cancer therapy by targeting hRad51, a key HR protein. hRad 51 has a unique activity: it promotes a search for homologous DNA sequences and DNA strand exchange between homologous DNA molecules, a basic step of HR. However, the mechanism of DNA strand exchange remains unknown. Specific inhibitors and stimulators of proteins are especially useful in determining the mechanism of enzymatic reactions.
Our goal is to identify specific modulators (inhibitors and stimulators), which can be used as chemical probes for analysis of the hRad51 mechanism and for development of novel anti-cancer therapies.

DNA strand exchange of hRad51 with fluorescently-labeled DNA substrates. To measure the hRad51 protein DNA strand exchange activity, a fluorimetric assay based on FRET was developed. In this assay, dsDNA substrate was prepared by annealing two complementary ssDNA oligonucleotides (47-mers): one containing fluorescein, a donor fluorophore with the excitation maximum at 490 nm and the emission maximum at 521 nm, at the 5'-end, and another containing black hole quencher 1 (BHQ1), a nonfluorescent acceptor, at the 3'-end. Since direct transfer of energy decreases with the sixth power of the distance between the fluorophores, the annealing of two complementary oligonucleotides increased direct energy transfer from donor to acceptor and thereby quenched the photon emission from the donor fluorescein group. The expected result of DNA strand exchange was an increase in fluorescence because displacement of the fluorescein carrying ssDNA strand from the duplex containing the quencher results in separation of the fluorescein and quencher groups. DNA strand exchange was initiated by addition of the dsDNA substrate to the hRad51 nucleoprotein filament that was formed on the non-fluorescent ssDNA identical in sequence to the fluorescein-labeled oligonucleotide. Inhibitors would be picked up as compounds that inhibit this fluorescence.

We have completed the HTS on ~200000 compounds (AID 1385). Hits identified were ordered from DPI and their dose-response is reported here.

Materials

Human Rad 51 protein, labeled ss & ds DNA were provided by the assay provider. The fluorescence assay was carried out in 384-well black, low-volume plates from Corning (Cat # 3676). All buffer salts were from Sigma.

Assay

hRad 51 protein was incubated with ss DNA to allow filament formation (37 deg C for 15 min). Compounds (8.5 uM final concentration) were pre-incubated with the protein for 30 min. followed by addition of ds DNA to initiate strand exchange. The reaction was allowed to proceed at room temperature for 15 min and then plates were read on Envision plate reader.


Dose response protocol

1.Serial dilute single compounds at in DMSO (16 two-fold dilutions from 2.5 mM to 75 nM)
2.Fill 384 well plate with 4 uL of water (nuclease-free) using Multidrop
3.Pin Tool compound into the plates using 384 pin-tool
4. Add 4 ul reaction mix (containing hRad 51 and ss DNA with buffers)
5.Preincubate compound with the reaction mix for 30 min at room temperature
6. Add 2 ul ds DNA
7.Incubate at room temperature for 15 min
8.Read fluorescence on Envision reader (Ex: 485 nm; Em: 520 nm)

Data analysis

IC50 plates contained compounds in columns 3-22, controls (DMSO, no compound) in columns 2 and 24, and blanks (heterologous DNA) in columns 1 and 23. Each column 3-22 contained 16 two-fold dilutions of a single compound, ranging in concentration from 85 uM to 2.5 nM. Percent activity was calculated for each dilution of each compound from the signal in fluorescence units (FU) and the mean of the plate controls and the mean of the plate blanks using the following equation:

% Activity = 100*((signal-blank mean)/(control mean-blank mean))

Dose response curves of percent activity were fit using XLfit equation 205 (four parameter logistic fit with maximum percent activity and minimum percent activity fixed at 100 and 0, respectively).

Activity scoring

The activity score reported here is based on follow-up IC50 testing on compounds that showed >40% inhibition in the primary HTS:

IC50 scores were calculated as follows:

(1) Score = 5.75 x (pIC50-3), where pIC50 = -log(10) of Mean IC50 in mol/L
(2) For IC50 >85 uM (or highest concentration tested), Score = 0

Activity Score Range:
For active compound, Score = 22-8
For Inactive compound, Score = 0

Activity Outcome

Compounds that gave percent inhibition >40 in the primary HTS were judged to be hits and these compounds were selected for follow-up IC50 testing. IC50 values were determined as described in protocol above.

Activity outcome is reported as follows:

(1) IC50 <85 uM in two or more IC50 determinations = active
(2) IC50 >85 uM (or highest concentration tested)= inactive

Contributors

This assay was submitted to the PCMD by Dr. Alex Mazin of Drexel University, assay development was done by Dr. Alex Mazin of Drexel University and Nuzhat Motlekar of UPenn, Dose response testing and data submission was done by Nuzhat Motlekar, of the University of Pennsylvania.

Correspondence

Please direct correspondence to Andrew Napper (napper@seas.upenn.edu).

Grant Number: MH084119