Inhibition of the BRCA2-RAD51 Interaction in Breast Cancer

Institution: University of California, Irvine
Investigator(s): Jiewen Zhu, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11FB-0117 Award: $134,769
Award Type: Postdoctoral Fellowship
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (2005)
BRCA2 was the second breast cancer susceptibility gene to be discovered, and it serves to repair DNA breaks along with another protein, called RAD51. The BRCA2-RAD51 interaction is essential for the DNA repair. In cancer cells, if this interaction is disrupted, the cell will become hypersensitive to DNA damage agent treatment. Therefore, this interaction may provide an ideal target for developing novel specific anti-cancer drugs.

Our preliminary studies have identified two small compounds, IBR1 and IBR2 that disrupt the BRCA2-Rad51 interaction. Furthermore, these compounds can inhibit breast cancer cell growth and induce hypersensitivity of breast cancer cells toward irradiation or cisplatin treatment. Therefore, we plan to modify these compounds and improve their effectiveness. First, a “library” of the small molecules will be designed and constructed by a combined approach of rational drug design and combinatorial synthesis. Then, a high-throughput screening for these second generation compounds will be carried out in pursuing more potent drug candidates. These successful candidates will be revalidated using various assays and a structure-activity relationship will be established. We plan to do much of our drug screening in yeast using the “two-hybrid” system. Then, we will screen active compounds for potency in cell growth assays. Finally, lead compounds will be tested for their synergistic effects with ionizing radiation and cisplatin.

Our primary goal is to modify the small compounds IBR1 and IBR2 so that they can disrupt the BRCA2-RAD51 interaction more efficiently. This may provide a new strategy in breast cancer treatment. Because IBR1 and IBR2 make the cancer cell hypersensitive to irradiation, a combinatory therapy with IBR1 or IBR2 can be developed, which will be more effective and tolerable than before.

Final Report (2008)
The BRCA2-RAD51 interaction is essential for the DNA repair. In cancer cells, if this interaction is disrupted, the cell will become hypersensitive to DNA damage agent treatment. Therefore, this interaction may provide an ideal target for developing novel specific anti-cancer drugs. IBR2 is a newly identified Rad51 inhibitor, which is able to disrupt Rad51-BRCA2 interaction and inhibit Rad51 self-multimerization in vitro, leading to the degradation of Rad51 and causing growth inhibition and cell death of breast cancer cells. The goal of the current project is to improve the efficacy as well as the pharmacological properties of IBR2.

In the previous reports, we have synthesized and screened IBR analogues for improved aqueous solubility; synthesized tumor-activated-prodrugs (TAP) of IBR2; synthesized and tested stabilized IBR2 analogues; developed a synthetic method for the two enantiomers of IBR-N; and designed a next generation Rad51 inhibitor by structure-based virtual ligand screening (VLS).

In the final project year, we stereo-selectively synthesized and tested the biological activity of a series of chiral derivatives; we have also explored the synthetic routes for several next generation compounds newly designed in silico; and we further consolidated the IBR2-Rad51 binding hypothesis using a rationally designed IBR2-conjugated affinity resin; lastly, we have determined the IBR2 pharmacokinetics properties.

In summary, we have modified IBR compounds, attempting to achieve better efficacy in inhibiting Rad51 and/or disrupting BRCA2/Rad51 interaction, resulting in inhibition of breast cancer cell growth, by applying a combined approach of computer-aided rational drug design / molecular modeling / virtual ligand screening and chemical synthesis. We have then performed biological function screenings and revalidated the IBR2 targeting model using chemical biological approaches. So far, the newly synthesized compounds haven’t exhibited significantly improved IC50; nevertheless, for certain compounds with similar IC50 as IBR2, improved solubility and/or stability has been identified. We have also been able to establish several synthetic routes, including two stereo-selective ones, which will be beneficial for future work. Our next steps of this project will be to continue the synthesis of a library of the newly designed Rad51 inhibitors, and to perform the biological screening of the resultant chemicals, in searching for a compound with better efficacy.

Symposium Abstract (2007)
The cytotoxicity of chemotherapy and radiation, two major treatments in advanced cancers, is directly related to an ability to cause DNA damage. The increased ability of cancer cells to recognize this damage and initiate DNA repair would have a negative impact upon therapeutic efficacy and result in resistance to these therapies. Therefore, the use of inhibitors of DNA repair pathways has the potential to enhance the cytotoxicity of genotoxic anticancer agents. Choosing Rad51 recombinase as a candidate is advantageous, because it is constantly overexpressed in cancer cells but not in normal somatic cells. Therefore, inhibition of Rad51-mediated pathways will have a much greater impact on the survival of the tumor cells than that of normal cells and appears to provide an exciting opportunity to selective killing of tumor cells. Previous studies have shown that inhibition of Rad51 function, by overexpressing exogenous BRC peptide, was able to arrest breast cancer cell growth and render cancer cells hypersensitive to ionizing radiation treatment. Therefore, the goal of this research project is to identify and modify small molecular compounds to effectively inhibit Rad51 recombinase. We expect these small molecules will provide a novel therapeutic treatment for breast cancer patients in near future.

Small molecular Rad51 inhibitors (IBR1/2) have been identified by a high-throughput reverse yeast-two-hybrid screening method from a library of 24,000 compounds. In vitro assays suggest that IBR2 binds to Rad51; and this binding interferes with its multimerization, a critical event for proper functioning of Rad51-mediated homologous recombination repair, as indicated by gel filtration assay. Molecular modeling studies, using DOCK or ICM software, suggest that IBR2 may structurally mimic a BRC motif in binding with Rad51. A series of IBR analogues were then synthesized and tested for their inhibitory effects on breast cancer cell growth; and structure-activity relationship analysis was performed using reverse yeast-two-hybrid assays and cell growth inhibition assays. The results suggested that the presence of a properly substituted phenyl moiety is essential for activity, which supports the current IBR2-Rad51 binding model. In IBR2-treated breast cancer cells, Rad51 became susceptible to degradation via proteosome-mediated pathway, and the growth rates of cancer cells were significantly reduced. IBR2 can also significantly retard xenografted breast tumor growth up to 60-70% at a dosage of 10-50 mg/kg in nude mice, without apparent general toxicity. Combination treatment of IBR2 with ionizing radiation enhanced its cytotoxic effect on tumor cells. These studies demonstrate a therapeutic potential of IBR molecules targeting Rad51 for degradation in breast cancer cells.

This project may provide new therapeutics in breast cancer treatment. First, IBR compounds alone can be used in inhibiting breast tumor growth. Second, since these compounds can enhance the sensitivity of breast cancer cells to irradiation, a combinatory therapy with lowered dosages can then be developed.