The reactivation of androgen receptor (AR) signaling in castration resistant prostate cancer (CRPC) in response to antiandrogen therapy renders the disease immune to available treatment options and is ultimately lethal. Currently, antiandrogen therapeutics block AR signaling by direct inhibition of androgen-AR interactions or indirectly through depletion of circulating androgens. The CRPC phenotype can arise from multiple cellular mechanisms including gain of function mutations to the AR gene or compensatory function by the glucocorticoid receptor (GR), another nuclear hormone receptor which binds to a nearly identical DNA response element as AR. While AR and GR signaling can be blocked by other DNA targeted therapeutics these compounds induce genotoxicity in tumor and healthy cells alike. The genotoxic effects in the tumor microenvironment often lead to a more aggressive treatment resistant phenotype. The Dervan laboratory has pioneered the development of Py-Im polyamides for targeting double- stranded DNA. These small molecules achieve affinities and specificities similar to DNA binding proteins, localize to the cell nucleus, bind chromatin, and modulate endogenous gene expression in cell culture. The minor groove binding Py-Im polyamides have been shown to inhibit both transcription factor-DNA binding (AR-ARE) and RNA polymerase II elongation without DNA cleavage. Our aims are focused on a series of Py-Im polyamides that bind the DNA sequence found within both canonical androgen and glucocorticoid response elements. The Py-Im polyamides will be examined against several models of prostate cancer that represent androgen insensitive disease. This project includes the study of prostate cancer xenografts in mice to determine the activity of polyamides against tumors derived from CRPC-relevant cell lines. Biodistribution analysis of recent polyamides with an optimized toxicity profile will be carried out in vivo using both pharmacokinetic analysis and dosimetry (14C label). We will investigate the in vivo chromatin binding of the polyamides using ChIP-Seq to validate the genomic binding profiles. Our long-term goal has been to develop a chemical method of modulating gene expression relevant for the treatment of cancer progression. The proposed research is significant because it will demonstrate that the mechanisms of resistance in prostate cancer can be attenuated with DNA-binding polyamides. Furthermore, this study will better our understanding of the effects of polyamide treatment against androgen insensitive prostate cancer in vivo, and contribute to the development of novel chemotherapeutic agents for the treatment of CRPC.