Journal Article

Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

Spies, M Ashley, Bain, Fletcher E, Malacaria, Eva, Hengel, Sarah R, Koch, Brandon G, Diaz, Andrea, Yu, Liping, Folly da Silva Constantino, Laura, Pichierri, Pietro, Spies, Maria, Wu, Meng

2016

Overview

The DNA repair protein RAD52 is an emerging therapeutic target of high importance for BRCA-deficient tumors. Depletion of RAD52 is synthetically lethal with defects in tumor suppressors BRCA1, BRCA2 and PALB2. RAD52 also participates in the recovery of the stalled replication forks. Anticipating that ssDNA binding activity underlies the RAD52 cellular functions, we carried out a high throughput screening campaign to identify compounds that disrupt the RAD52-ssDNA interaction. Lead compounds were confirmed as RAD52 inhibitors in biochemical assays. Computational analysis predicted that these inhibitors bind within the ssDNA-binding groove of the RAD52 oligomeric ring. The nature of the inhibitor-RAD52 complex was validated through an in silico screening campaign, culminating in the discovery of an additional RAD52 inhibitor. Cellular studies with our inhibitors showed that the RAD52-ssDNA interaction enables its function at stalled replication forks, and that the inhibition of RAD52-ssDNA binding acts additively with BRCA2 or MUS81 depletion in cell killing. Cells are constantly in danger of losing or scrambling critical genetic information because of DNA damage. To cope with this stress, cells have numerous DNA repair systems. One of these systems – homology-directed DNA repair – involves the proteins BRCA1 and BRCA2, which are often missing or defective in breast and ovarian cancers. The BRCA-deficient cancer cells can still survive, but become “addicted” to other DNA repair proteins – among them a protein called RAD52. It might be possible to kill these cancer cells using drugs that stop RAD52 from working. Such treatments would have the benefit of not harming normal healthy cells, as these cells contain working BRCA proteins and can survive without RAD52. It is not currently known exactly how RAD52 allows the BRCA-deficient cells to survive, but this probably depends on RAD52’s ability to bind to single strands of DNA. Small molecules that block the interaction between the RAD52 protein and DNA might therefore help to kill cancer cells. Hengel et al. developed a high throughput biophysical method to search through a large collection of small molecules to find those that prevent RAD52 from binding to DNA. The best potential drug leads were then tested in laboratory-grown human cells and using biophysical and biochemical techniques. Computational approaches were also used to model how these molecules block the interaction between RAD52 and DNA at the atomistic level. Hengel et al. then used the information about how the small molecules bind to RAD52 to perform further computational screening. This identified a natural compound that competes with single-stranded DNA to bind to RAD52. The activity of this molecule was then validated using biophysical methods. The methods used by Hengel et al. provide the foundation for further searches for new anticancer drugs. Future studies that employ the small molecule drugs identified so far will also help to determine exactly how RAD52 works in human cells and how it helps cancer cells to survive.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Anemia / Biochemistry / BRCA1 protein / BRCA2 / BRCA2 protein / BRCA2 Protein - deficiency / Cell Line, Tumor / Cell Survival - drug effects / Computer applications / Deoxyribonucleic acid / DNA / DNA repair / DNA, Single-Stranded - metabolism / Enzyme Inhibitors - isolation & purification / Enzyme Inhibitors - metabolism / Fibroblasts - drug effects / Fibroblasts - physiology / Genes and Chromosomes / High-throughput screening / Humans / MUS81 / Natural products / Protein Binding - drug effects / Proteins / RAD52 / Rad52 DNA Repair and Recombination Protein - metabolism / Rad52 protein / Recovery (Medical) / Replication forks / small-molecule inhibitors / Tumors