Homologous recombination - Rad 51
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 more ..
BioActive Compounds: 169
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.
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.
hRad 51 protein was incubated with ss DNA to allow filament formation (37 deg C for 15 min). Compounds (10 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.
1.Fill 384 well plate with 4 uL of water (nuclease-free) using Multidrop
2.Pin Tool compound into the plates using 384 pin-tool
3. Add 4 ul reaction mix (containing hRad 51 and ss DNA with buffers)
4.Preincubate compound with the reaction mix for 30 min at room temperature
5. Add 2 ul ds DNA
6.Incubate at room temperature for 15 min
7.Read fluorescence on Envision reader (Ex: 485 nm; Em: 520 nm)
The data was analyzed in IDBS Activity Base XE. Each HTS plate had a single test compound (10 uM in 1.7% DMSO) in columns 3-22, controls (1.7% DMSO) in columns 2 and 24, and blanks (heterologous ss DNA) in columns 1 and 23. Percent inhibition was calculated for 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:
% Inhibition = 100*(1-((signal-blank mean)/(control mean-blank mean)))
For positive percent inhibition, score = 0.4 x percent inhibition
For negative percent inhibition, score = 0
(Note that compounds that fluoresce at excitation 485/emission 520 nm give a signal higher than the plate control that corresponds to a substantial negative percent inhibition)
Results of screening the MLSCN library of compounds against hRad 51 were as follows:
Total no. of compounds tested: 198,479
Compounds that gave percent inhibition >40 at 10 uM concentration (Hits): 169
Compounds that gave percent inhibition <40 at 10 uM concentration (Inactive): 198,310
The hits will be retested in a dose-response assay for confirmation of activity.
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, HTS was conducted by Nuzhat Motlekar and Parul Jhunjhunwala, and data was submitted by Nuzhat Motlekar, of the University of Pennsylvania.
Please direct correspondence to Andrew Napper (firstname.lastname@example.org).
** Test Concentration.
Data Table (Concise)