Small Molecule Therapeutics Activity of a Py–Im Polyamide Targeted to the Estrogen Response Element

Pyrrole-imidazole (Py–Im) polyamides are a class of programmable DNAminor groove binders capable of modulating the activity of DNA-binding proteins and affecting changes in gene expression. Estrogen receptor alpha (ERa) is a ligand-activated hormone receptor that binds as a homodimer to estrogen response elements (ERE) and is a driving oncogene in amajority of breast cancers.We tested a selection of structurally similar Py– Im polyamides with differing DNA sequence specificity for activity against 17b-estadiol (E2)–induced transcription and cytotoxicity in ERa positive, E2-stimulated T47DKBluc cells, which express luciferase under ERa control. The most active polyamide targeted the sequence 50-WGGWCW-30 (W 1⁄4 A or T), which is the canonical ERE half site. Whole transcriptome analysis using RNA-Seq revealed that treatment of E2stimulated breast cancer cells with this polyamide reduced the effects of E2 on the majority of those most strongly affected by E2 but had much less effect on the majority of E2-induced transcripts. In vivo, this polyamide circulated at detectable levels following subcutaneous injection and reduced levels of ER-driven luciferase expression in xenografted tumors in mice after subcutaneous compound administration without significant host toxicity. Mol Cancer Ther; 12(5); 675–84. 2013 AACR. Introduction Estrogen receptor-alpha (ERa) is a member of the nuclear hormone receptor family of transcription factors and is active in amajority of breast adenocarcinomas (1, 2). Breast tumors that express ERa and are sensitive to circulating estrogens respond to therapeutics that modulate ERa activity (3). Such therapeutics include tamoxifen, a selective ERmodulator that acts as aweak agonist/ antagonist by binding to the ERa ligand-binding pocket and the aromatase inhibitors that inhibit synthesis of E2 (3). A different strategy for modulation of ERa activity is inhibition of the ERa–ERE interface by a DNA-binding molecule. Pyrrole-imidazole (Py–Im) polyamides are a class of synthetic, minor groove-binding ligands inspired by the natural product distamycin A (4, 5). Py–Im polyamides are oligomers of aromatic amino acids linked in series to fold in an antiparallel fashion when bound in the minor groove of DNA (4, 5). Sequence specificity is programmed through side-by-side pairings of the Py and Im subunits that recognize differences in the shape and hydrogen bonding pattern presented by the edges of the Watson– Crick base pairs in the floor of the minor groove (6, 7). Binding specificity has been extensively characterized by DNAse I footprinting titrations and other methods. An Im:Py pair preferentially recognizes G:C; Py:Im prefers C: G, and Py:Py is degenerate for A:T and T:A (6, 7). Py–Im polyamide binding in the minor groove also induces allosteric changes to the major groove (8, 9) and binding affinity is sufficient to modulate the binding of DNAbinding proteins (8–11). In cell culture, selected polyamides have been used to modulate expression of genes induced by testosterone (11), TNF-a (12), hypoxia (13, 14), and dexamethasone (10). The mechanisms by which polyamides affect gene expression changes in cell culture is still not well understood and may involve direct effects on multiple DNAdependent processes including transcription factor occupancy, chromatin structure, RNApolymerases, and DNA replication (15). The pharmacokinetics and toxicity of a number of polyamides after intravenous and subcutaneous injection inmice and rats have beendescribed (16–18). In mice, a selected polyamide was reported to induce changes in TGF-b expression in kidney glomeruli, and a fluorescent analog of this polyamide was observed in kidney glomeruli after tail vein injection in rats (19). Gene expression changes have also been observed in tumor Authors' Affiliations: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena; and Department of Radiation Oncology, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). N.G. Nickols and J.O. Szablowski contributed equally to this work. Corresponding Author: Peter B. Dervan, Division of Chemistry and Chemical Engineering, California Institute of Technology, l64-30, Pasadena, CA 91125. Phone: 626-395-6002; Fax: 626-683-8753; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-12-104