ER has emerged as one of the most effective targets for breast cancer therapy since the discovery of the estrogen receptor (ER) as the modulator of estrogen action. However, the current ER-targeting therapies have a main limitation related to resistance. Our previous studies have shown that XR5944, a DNA bis-intercalator is capable of inhibiting the ER activity by its ability to specifically block the binding of ER to its consensus EREs. An DNA intercalator that targets ERE may be a useful therapeutic agent that can overcome the resistance to existing endocrine therapies. To understand the mechanism of the inhibition of ER-ERE interactions by XR5944, we have tested the specificity of XR5944 binding to consensus EREs (AGGTCAnnnTGACCT) with different tri-nucleotide spacer sequences as well as natural EREs using 1H NMR titration studies. Our results suggested that the ERE spacers play a significant role in determining the binding characteristics of the complex. XR5944 binds the ERE sequences in a slow-exchange binding mode on the NMR time-scale. The binding stoichiometry of XR5944 with ERE sequences was shown to be 2:1. 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. 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. Natural ERE sequences, including those located in the promoter of ER target genes TFF1, GREB1, Cathepsin D, Lactoferrin, and TGF-α, also appear to bind to XR5944 with different specificities as evident by differences in the 1H NMR spectra of their drug-DNA complexes. Thus, the affinity and specificity of the XR5944-ERE complex is influenced both by the nucleotide sequence of the half-sties and the tri-nucleotide spacer. Of the natural EREs tested, XR5944 appears to bind with the highest specificity to the ERE sequence of TFF1; hence we carried out further NMR studies of the binding of XR5944 to the TFF1 ERE sequence. Using site-specific labeling at each base, we were able to assign both the imino and H8 protons of the free TFF1 DNA and its drug complex. These assignments provide important information on the XR5944 binding sites with TFF1 ERE.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4514. doi:10.1158/1538-7445.AM2011-4514