Graduation Date

Spring 5-5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Dr. Gloria Borgstahl

Abstract

Germline BRCA mutations underlay a significant risk for breast and ovarian cancer that increases in age. BRCA mutations are usually associated with the most aggressive subtypes of these cancers such as triple negative breast cancer and high-grade serous ovarian cancer. Conventional chemotherapeutic or hormonal therapies do not address the molecular deficiencies responsible for their resistance and there is a high rate of recurrence. Targeted therapy that can address the unique molecular features in these subtypes of cancer is the only way to cure the disease or, at the very least, improve patients’ quality of life.

Homologous recombination repair is an accurate repair pathway that utilizes a copy of a homologous sequence to relay information to the break site. Cancer cells copy their DNA extensively meeting the principle demand for this high-fidelity repair pathway. Homologous recombination repair is utilized by cancer cells to cope with the most challenging forms of DNA damage such as DNA double strand breaks, stalled replication forks, adducts, and interstrand crosslinks. Among the key proteins in homologous recombination repair, RAD52 activity promotes cancer cells’ tolerance and survival. Therefore, there is a therapeutic opportunity in inhibiting RAD52 activity to push DNA damage levels in homologous recombination repair-deficient tumors beyond the limits of viability.

One of the early events in this repair is resection of the broken strand and generation of single strand DNA. Replication protein A cover and protect those strands and interact with key DNA repair proteins. RAD52 activity in DNA repair is dependent on its interaction with replication protein A. The hypothesis of this thesis is that it is possible to inhibit RAD52 activity by inhibiting its interaction with RPA and this inhibition will have therapeutic benefits for cancer patients. We explored the binding activity and affinity of the RAD52 interaction with RPA. Kinetic, and thermodynamic parameters dictating this protein:protein interaction were measured. The characterization of RAD52:RPA interaction data guided remapping of the RPA interaction domains on RAD52. To target RAD52 activity by inhibiting its interaction with RPA, we designed an in vitro fluorescent-based protein-protein interaction assay. This assay was further optimized for high throughput settings with a robust signal, minimal steps, statistical accuracy, and low cost. We screened over 100,000 compounds to look for small molecule inhibitors. Eleven hits were found and five were selected for their high EC50 values. Three of the five hits are FDA approved drugs and were selected for cytotoxicity tests in BRCA-deficient cell lines.

The outcome of our characterization for these three candidate small molecule inhibitors may shed light on the variation of their efficacy and sensitivity among breast or ovarian cancer patients with BRCA-defective pathway versus those with none. Additionally, we present fluorescent-based protein-protein interaction assay as an affordable method to detect many protein:protein interactions in low-scale or high throughput settings applicable to finding small molecule inhibitors or aptamer modulators for protein:protein interactions.

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