DNA is a major target for drugs used in cancer therapy. However, DNA interactive chemotherapeutics are limited by adverse effects due to their poor selectivity. The bis-phenazine compound XR5944 is a DNA bis-intercalator and potent anticancer drug which reached phase I clinical trials. The DNA binding mode and mechanism of XR5944 are unique, allowing it to recognize and bind the estrogen response element (ERE) sequence to inhibit ERα activity and making it highly effective against ER-positive breast cancer in vitro. Understanding XR5944's novel mechanism of DNA recognition may allow the further development of anti-cancer agents capable of overcoming drug resistance to current anti-estrogen treatments by directly targeting transcriptional control, bypassing the hormone-receptor complex currently targeted by anti-estrogen treatments. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the structure of XR5944 in complex with its preferred DNA duplex sequence identified its binding mode. The structure of the 2:1 complex of XR5944 with the naturally occurring TFF1-ERE exhibits several unexpected features. Our NMR structure shows the two phenazine moieties are well stacked and the carboxamide amino linker lies in the major groove of DNA. At both binding sites in the 2:1 complex, XR5944 intercalates strongly at one site but weakly at the other. The binding sites within a native promoter sequence appear to be context- and sequence- dependent. Taken together, our results highlight that the DNA binding of a bis-intercalator is different from the simple addition of two single intercalation sites. Our study suggests that improved XR5944 derivatives targeting EREs may be designed through optimization of aminoalkyl linker and intercalation moieties.

Citation Format: Clement Lin, Raveendra I. Mathad, Neil Sidell, Danzhou Yang. DNA recognition by XR5944, a novel bis-intercalator and potent anticancer drug. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2445. doi:10.1158/1538-7445.AM2015-2445