Dissertation
Therapeutic disruption of RAD52-ssDNA complexation using novel drug-like scaffolds
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2023
DOI: 10.25820/etd.007156
Abstract
DNA repair protein RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers such as breast cancer susceptibility gene 1 (BRCA1) and breast cancer susceptibility gene 2 (BRCA2) (~25% of breast and ovarian cancers). The genome stabilizing functions of RAD52 that underlies its use in cells displaying so-called BRCAness phenotype depends on the ability of RAD52 to bind ssDNA. However, there remains a dearth of small molecule inhibitors that can abrogate the interaction between ssDNA and RAD52. This is because structure activity relationships for RAD52 are complex which makes it challenging to transform previously identified natural product and repurposed compounds that inhibit the RAD52-ssDNA interaction into drug like compounds with good ADME qualities that are suitable for clinical use.
Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we have identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. I performed FRET-based assays to characterize these six compounds and confirm their ability to act as RAD52 inhibitors. All six compounds were shown to inhibit the complexation between ssDNA and RAD52 (IC50 ~23-1200 μM). Moreover, two of the compounds (Z56 and Z99) were capable of selectively killing BRCA1 and BRCA2 mutated cells. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA2-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA2-complemented cells as well as BRCA proficient cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ~1.3-8 μM), which were only toxic to BRCA1 and BRCA2-mutant cells.
Moreover, our RAD52 inhibitors could also affect the proliferation and clonogenic potential of glioblastoma cells that don’t have mutations in BRCA1 and BRCA2. This data suggests that RAD52 plays a role in the survival of glioblastoma cells and provides impetus for the expansion of RAD52 inhibitor treatment into other types of cancers outside of BRCA-deficient breast, ovarian and pancreatic malignancies. Thus, RAD52 complexation by Z56, Z99 and its more specific derivatives provides a roadmap for further improvement of our lead compounds.
Details
- Title: Subtitle
- Therapeutic disruption of RAD52-ssDNA complexation using novel drug-like scaffolds
- Creators
- Divya S Bhat
- Contributors
- Maria Spies (Advisor)Adam Dupuy (Committee Member)Pietro Pichierri (Committee Member)Michael Henry (Committee Member)Sarit Smolikove (Committee Member)Anna Malkova (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biomedical Science (Cancer Biology)
- Date degree season
- Spring 2023
- DOI
- 10.25820/etd.007156
- Publisher
- University of Iowa
- Number of pages
- xvi, 137 pages
- Copyright
- Copyright 2023 Divya S Bhat
- Language
- English
- Date submitted
- 04/24/2023
- Date approved
- 05/24/2023
- Description illustrations
- Illustrations, tables, graphs, charts
- Description bibliographic
- Includes bibliographical references (pages 124-137).
- Public Abstract (ETD)
Defects in DNA repair proteins BRCA1 and BRCA2 cause the development of breast and ovarian cancers. The current treatments for these types of cancers are surgical intervention, prophylactic surgeries (for carriers of pathogenic mutations) radiation and chemotherapy. Studies show that cancer cells that have defects in BRCA1 and/or BRCA2 depend on other DNA repair proteins for survival, treatment resistance and uncontrolled growth. RAD52, a DNA repair protein has been shown to aid BRCA deficient cells in managing DNA repair. Thus, if one depletes or inhibits RAD52, one can kill BRCA-deficient cancerous cells with lesser or no negative effects on normal non-diseased cells. The challenge in clinical targeting of RAD52 is a lack of drug like compounds that can inhibit its activity as a DNA repair protein. My study has designed six novel compounds from a virtual chemical library and characterized them as RAD52 inhibitors. Two of these compounds (Z56 and Z99) were capable of selectively inhibiting the growth of BRCA mutated cancer cells. However, we realized that the efficacy and specificity of these compounds was low and could be improved upon.
Using a secondary computational method, we were able to modify the structure of one of our RAD52 inhibitor compounds and in the process found six compounds that had an improved efficacy for targeting RAD52 DNA repair functions. These compounds were shown to be highly specific in targeting BRCA-mutated cells. Upon testing these compounds in brain cancer (glioblastoma) cell lines we were surprised to find that RAD52 also plays a role in maintaining the growth of brain cancer cells. While the functions of RAD52 in aiding the survival of BRCA deficient and brain cancer cells is still unknown, our data provides a set a molecular tools that can not only be used to determine the role of RAD52 in aiding cancer cell DNA repair, but also can be used as an effective anti-cancer treatment in the future.
- Academic Unit
- Biomedical Science Program
- Record Identifier
- 9984424791402771
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