Hormone therapies interfere with estrogen signaling by targeting Estrogen Receptors (ERs) and are routinely used for the prevention and treatment of ER-expressing breast cancers. However, current therapies have significant limitations related to inherent or acquired resistance. Thus, there is a need to develop therapeutic compounds that do not depend on competitive binding to the ER or on depleting cells of endogenous estrogen. Previous data suggested that certain bis-phenazines, such as our lead compound XR5944, possess potent antiestrogenic properties through a novel mechanism of action: they inhibit gene transcription via direct intercalation with DNA at estrogen response elements (EREs) which are located in estrogen-responsive gene promoters. While these studies have demonstrated the potent antiestrogenic activity of XR5944, they have also demonstrated its capacity to bind to alternative DNA motifs. In order to improve upon ERE target specificity, we have identified XR5944 derivatives that retain potent antiestrogenic properties but may have greater specificity for ERE sequences, to be developed as new therapies for the treatment of estrogen-driven cancers. Such compounds may also be effective in overcoming resistance to current antiestrogen therapies. Here, we characterize DNA sequence interactions and transcriptional effects of five XR5944 derivatives that differ from the parent compound in the composition of their linker chains (compounds 26357 and 26373), cyclic side rings (26576), or both (26536 and 27402). We utilized 1D 1H NMR titration experiments to monitor compound:DNA binding properties to a canonical ERE (AGGTCAcggTGACCT) containing a CGG trinucleotide spacer (ERECGG), electrophoretic mobility shift assays (EMSA) to assess the ability of the derivatives to disrupt ER:ERECGG interactions, and luciferase reporter assays to evaluate their potency in blocking estrogen-dependent transactivation through ERECGG. Our NMR results indicated that alteration of either linker chain or side ring structures of XR5944 reduced the affinity and specificity of compound:ERECGG interactions. These findings correlated with the ability of the compounds to inhibit ER:ERECGG binding in the order of XR5944>26576>26536>26373>26357>27402, as evaluated by EMSA. To further determine the effects of the compounds on ERECGG-driven transactivation, we performed luciferase reporter assays utilizing ERα-expressing MCF-7 breast cancer cells transfected with an ERECGG-luciferase reporter construct. Unlike the EMSA results, there was not a strict concordance between the compound-dependent potency in inhibiting estrogen-driven transcriptional responses and the specificity of interaction with DNA as determined by NMR. Specifically, inhibition of reporter activity was more potent with compounds that had the same cyclic side rings but altered linker chains as XR5944 (26373 and 26357) and was less effective with compounds possessing altered side rings (26576, 26536, and 27402). Together, our findings indicate that the cyclic side chain structures appear to be the most important components of bis-phenazines in disrupting the transcriptional potential of ER:ERE interactions. NMR can be useful for predicting the relative potency of XR5944 derivatives in blocking ER binding to EREs in vitro, but fails to predict their efficacy in inhibiting ERE-mediated transactivation. This information should prove valuable for the rational drug design of new antiestrogenic compounds with improved specificity for ERE sequences.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B66.