Since the discovery of the estrogen receptor (ER) as the modulator of estrogen action, ER has evolved to be one of the most effective targets for breast cancer therapy. However, the current ER-targeting therapies have a main limitation related to resistance. Our previous studies have shown that XR5944, a DNA bis-intercalator with potent anticancer activity, is capable of inhibiting the ER activity by its ability to specifically block the binding of ER to its consensus DNA sequence ERE compared to other transcription factor-target DNA interactions. This novel mechanism of ER inhibition by a DNA bis-intercalator may be useful in overcoming resistance to current antiestrogens. The consensus ERE is an inverted repeat comprised of two ERE half-sites separated by three bases: AGGTCAnnnTGACCT where nnn is known as the tri-nucleotide spacer. ER can also bind to non-consensus EREs, i.e. natural EREs that differ from the consensus ERE by one or more base pairs but nevertheless serve as potent transactivation sites for estrogen target genes Therefore, to further understand the mechanism and specificity of the inhibition of ER by XR5944, we tested the specificity of XR5944 to block interactions with consensus and non-consensus ERE sequences by 1D 1H NMR titration studies. Our NMR studies of XR5944-DNA interactions suggested that the consensus ERE spacer plays a significant role in determining the binding characteristics of the complex. Of those tested, the CGG/GCC and AGG/CCT spacers showed the best binding, while the CGT/ACG and TTT/AAA spacers showed the worst binding with XR5944. The binding stoichiometry of XR5944 with ERE sequences appears to be 2:1, which explains why the spacer region can affect the drug-DNA binding. To validate our NMR results, we have also conducted functional studies by using reporter constructs containing EREs with tri-nucleotide spacers CGG and TTT. Results of luciferase reporter assays in MCF-7 cells indicated that XR5944 was significantly more potent in inhibiting the activity of reporters containing ERE-CGG than those containing ERE-TTT, consistent with our NMR results. Together with our NMR studies, these reporter gene experiments indicate that the tri-nucleotide spacer plays a significant role in the binding characteristics and functional consequence of XR5944 interactions with the consensus ERE.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4616.