Genomic DNA is the predominant target of many chemo- and radio- therapies, however these therapies are genotoxic and non-specific. Small molecules that interact with DNA without genotoxicity and with sequence specificity could be a significant advance over conventional DNA-targeted therapeutics. Pyrrole-Imidazole (Py- Im) polyamides are a class of non-covalent DNA-binding small molecules with programmable sequence recognition and high affinity. Sequence preference is directed by side-by-side pairings of aromatic amino acids in the minor groove that can distinguish the four Watson-Crick base pairs. Polyamide-DNA binding does not result in genotoxicity and sequence specificity is programmable. This distinguishes these molecules from conventional DNA-targeted therapeutics. As a research-track resident and visiting postdoctoral researcher in Peter Dervan's laboratory, I used this technology platform to target transcription in prostate cancer. He developed eight-ring hairpin-polyamides that target the Androgen Receptor (AR) AR response element (ARE) sequence 5'-WGWWCW-3' (W=A or T). These polyamides reduce occupancy of AR in nuclear chromatin, transcription by RNA Polymerase II (RNAP2), and strongly suppress castrate-sensitive prostate cancer xenografts with minimal systemic toxicity in mice. This work has given rise to three related hypotheses that form the basis for the research strategy of this CDA-2 proposal: (1) Because ARE-targeted polyamides interfere with AR-ARE binding by targeting the DNA rather than the ligand binding domain of AR, these molecules should retain efficacy against castrate-resistant prostate tumors that over-express AR, and castrate resistant prostate tumors with AR splice variants which lack the ligand binding domain and are resistant to all clinically used anti-androgens (including second- generation anti-androgens like enzalutamide). (2) Because polyamide-DNA binding interferes with AR-ARE binding, we hypothesize that polyamides can be used to modulate AR-directed chromatin structure in prostate cancer cells. This is important because AR-directed chromatin structure, in concert with ionizing radiation, influences the formation of additional genetic changes (such as formation of the TMPRSS2-ERG translocation) that are drivers of prostate cancer progression. (3) Because RNAP2 is required for DNA damage repair, and polyamide-treatment transiently decreases levels of this protein, we hypothesize that polyamides may sensitize cells to ionizing radiation. These hypotheses are explored Aims 1, 2, and 3, respectively. My mentors are all investigators with proven track records in the mentorship of academicians. My primary mentor, Matthew Rettig MD, is chief of hematology-oncology at the West LA VA and leads a productive, well- funded basic and translational research lab focused on prostate cancer. His co-mentors Peter Dervan (chemical biology) and William McBride (radiobiology) are renown in their respective fields. The proposal builds upon the applicants' prior work with co-mentor Peter Dervan PhD but expands into new areas - clinically relevant applications in advanced prostate cancer, justifying primary mentorship with Matthew Rettig MD, and in radiobiology, justifying co-mentorship with William McBride PhD. I have explicit support by his mentors in carrying forth these research topics into his independent career. The overall goal for the field of Py-Im polyamides is clinical application for advanced prostate cancer, which is a highly relevant goal for the health of American Veterans.