Abstract #5545: The effect of a small molecule inhibitor of Replication Protein A (TDRL-505) on DNA binding, cellular function and platinum sensitivity (2009)

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

Replication protein A (RPA) is the eukaryotic single-strand DNA binding protein and is involved in several DNA metabolic pathways. RPA is a critical component of the nucleotide excision repair (NER) pathway which is responsible for repair of bulky DNA adducts including those induced by cisplatin, a common chemotherapeutic agent used for treating various types of cancer. RPA is also indispensible for DNA replication and recombination. RPA\#8217;s participation in these pathways is due to its high affinity DNA binding activity. The central role of RPA in several DNA metabolic pathways makes it an attractive target for inhibition by small molecules both as a novel chemotherapeutic target and as a means of increasing cisplatin sensitivity. Using a novel high-throughput screening assay, we have identified a series of structurally similar small molecules that inhibit the DNA binding activity of RPA. Using this core structure, we systematically identified and screened an additional series of compounds. We present the characterization of one compound identified in this series, TDRL-505, that has both in vitro and biological activity consistent with inhibition of RPA. In vitro inhibition of RPA binding was observed in electrophoretic mobility shift and fluorescence polarization assays. In order to determine the effect of RPA inhibition on biologic activity, TDRL-505 was assessed in H460 non-small cell lung cancer cells. The ability of TDRL-505 to induce cell killing was analyzed using the Annexin V/propidium iodide flow cytometric assay as well as in a crystal violet-based cell viability assay. TDRL-505 was found to induce cell killing in both assays with an IC50 value of ~45 µM. To determine the potential mechanism of cytotoxicity, cell cycle analysis of the inhibitor in H460 cells was examined using propidium iodide DNA staining. Cells treated with compound showed an arrest in the G1 phase of the cell cycle, consistent with inhibition of RPA\#8217;s role in S-phase DNA replication. Cells synchronized in G2/M with nocodazole were also analyzed for the ability of the compound to prevent entry into S-phase. After treatment with nocodazole, cells released from nocodazole arrest and treated with TDRL-505 were unable to exit G1 and enter S-phase, again consistent with inhibition of cellular RPA activity. In order to determine if the compound affected RPA\#8217;s role in DNA repair, we examined its activity in conjunction with cisplatin treatment of H460 cells. This study showed a greater than additive increase in cell death with both the RPA inhibitor and cisplatin compared to either agent alone. Combination index analysis demonstrated synergistic activity between the two compounds. These results demonstrate that a small molecule inhibitor of RPA\#8217;s in vitro DNA binding activity has cellular activity consistent with inhibition of RPA\#8217;s role in both DNA replication and repair in vivo .

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5545.