Mona Al-Mugotir (University of Nebraska Medical Center)| Carol Kolar (University of Nebraska Medical Center)| David Kelly (University of Nebraska Medical Center)| Bessho Tadayoshi (University of Nebraska Medical Center)| Amarnath Natarajan (University of Nebraska Medical Center)| Gloria Borgstahl (University of Nebraska Medical Center)
Malignancies are characterized by high levels of DNA damage and double-strand break (DSB) is the most toxic and challenging to repair. Chemotherapeutics and radiation drive further DNA damage raising the demand for DNA repair. The homologous recombination (HR) is most significant in DSB repair. Single-stranded DNA (ssDNA) resulting from the initial processing of the damage and awaiting recruitment of repair proteins are protected by the binding of replication protein A (RPA). Subsequent HR elements, BRCA1, BRCA2, and PALB2 proteins, mediate crucial steps in the repair and, not surprisingly, cause some of the most aggressive subtypes of familial cancers when mutated on the gene level. In such cases, a protein called RAD52, which is the main player in single-strand annealing (SSA), leads a backup pathway for HR repair. For this reason, inhibition of RAD52 activity presents a synthetic lethality therapeutic opportunity . Additional reports in recent years demonstrated evidence to support various roles of RAD52 in promoting tumorigenesis . This work aims to target RAD52 by inhibiting its protein-protein interaction (PPI) with RPA. Preceding work demonstrated that RAD52 activity and subsequent repair steps are crucially dependent on interaction with RPA . Methods included an ELISA-based high throughput assay screen (HTS) developed especially for this project allowed the selection of 11 from over 100,000 small molecule inhibitors (SMIs) screened. A hit was identified for its ability to inhibit the PPI statistically. A special effort was placed on three of the hits being FDA-approved drugs. Accordingly, their effect on BRCA1/2-deficient human cancer cell lines' survival was investigated. All SMIs were characterized by biophysical techniques to identify protein binding partners and kinetic parameters of the interaction respectfully. Current results suggest interaction with RAD52 at a micromolar level. The outcome of this work includes the design and development of PPI applicable in low and high throughput settings , characterization of previously undefined PPI parameters, and preliminary evidence for this PPI complex being a cancer drug target. The latter effort is being expanded to investigate a potential new mechanism-of-action for a drug in clinical use which may have significance in supporting efforts toward personalized medicine.
1. Lok, B.H., et al., RAD52 inactivation is synthetically lethal with deficiencies in BRCA1 and PALB2 in addition to BRCA2 through RAD51-mediated homologous recombination. Oncogene, 2013. 32(30): p. 3552-8.
2. Lieberman, R., et al., Rad52 deficiency decreases development of lung squamous cell carcinomas by enhancing immuno-surveillance. Oncotarget, 2017. 8(21): p. 34032-34044.
3. Deng, X., et al., Human replication protein A-Rad52-single-stranded DNA complex: stoichiometry and evidence for strand transfer regulation by phosphorylation. Biochemistry, 2009. 48(28): p. 6633-43.
4. Al-Mugotir, M., et al., A simple fluorescent assay for the discovery of protein-protein interaction inhibitors. Anal Biochem, 2019.