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Dissertation
Targeting RAD52 using small-molecule inhibitor and understanding its essential role in BRCA-deficient cells
Doctor of Philosophy (Ph.D.), Drexel University
Jul 2020
DOI:
https://doi.org/10.17918/00000295
Abstract
RAD52 is a member of the homologous recombination pathway that is important for survival of BRCA-deficient cells. Disfunction of RAD52 leads to lethality in BRCA-deficient cells. However, the exact mechanism of how RAD52 contributes to viability of BRCA-deficient cells remains unknown. Two major activities of RAD52 were previously identified: DNA or RNA pairing, which includes DNA/RNA annealing and strand exchange, and mediator, which is to assist RAD51 loading on RPA-covered ssDNA. Here, we report that the N-terminal domain (NTD) of RAD52 devoid of the potential mediator function is essential for maintaining viability of BRCA-deficient cells owing to its ability to promote DNA/RNA pairing. We show that RAD52 NTD forms nuclear foci upon DNA damage in BRCA-deficient human cells and promotes DNA double-strand break repair through two pathways: homology-directed repair (HDR) and single-strand annealing (SSA). Furthermore, we show that mutations in the RAD52 NTD that disrupt these activities fail to maintain viability of BRCA-deficient cells. We also explored RAD52 as a potential therapeutic target to selectively eliminate BRCA-deficient cells with a novel small-molecule inhibitor. We screened a compound library of 372,903 compounds and identified D-I03 as the lead compound showing strongest effect in inhibiting RAD52 in vitro and in vivo both as a single agent and in combination with PARP inhibitors, which were recently approved for treatment BRCA-deficient ovarian cancer. Given the promising therapeutic potential of inhibiting RAD52 by D-I03, structure-activity relationship studies were conducted which identified 63 proprietary compounds more potent than D-I03 in inhibiting RAD52. We tested these compounds for anti-proliferating activity against BRCA-deficient cancer cells in vitro and identified 3 best RAD52i for in vivo studies. Targeting RAD52 with novel small-molecule inhibitor and understanding its functional role would help us elucidate the mechanism of DNA repair in BRCA-deficient cancer cells and hence allow efficient targeting.
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Details
- Title
- Targeting RAD52 using small-molecule inhibitor and understanding its essential role in BRCA-deficient cells
- Creators
- Kritika Hanamshet
- Contributors
- Alexander V. Mazin (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xiii, 148 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991014695135804721