Abstract Replication Protein A (RPA) is a major regulator of checkpoint activation and enhanced DNA repair in cancer cells. In response to genotoxic stress, the RPA complex binds to and protects ssDNA while serving as a scaffold to recruit critical checkpoint and DNA-damage response proteins through the N-terminal region of the 70 kDa subunit of RPA (RPA70N). Specific disruption of the protein-protein interactions mediated by the RPA70N domain has the potential to produce selective killing of cancer cells without the risk of cytotoxicity due to interference in the ssDNA-binding function. Stapled helix peptides can serve as useful tools for inhibiting protein-protein interactions. However, their utility can be limited due to difficulties often encountered during attempts to improve the binding affinity to the target. Here, we report the discovery and optimization of a potent stapled helix peptide probe, derived from the endogenous RPA binding partner ATRIP (ATR-interacting protein), that binds to and inhibits the RPA70N protein-protein interaction surface. Alanine scanning, charge abrogation, and rational sequence optimization resulted in a peptide with a 100-fold potency gain over the native sequence and improved physical characteristics. In addition to the application of these traditional strategies, we describe a novel approach for efficiently designing peptides containing unnatural amino acids. This method involves the incorporation of an unnatural amino acid inspired by the structure activity relationships of small molecules that bind to the same site on the protein. Use of this approach produced stapled peptides with dramatic increases in binding affinity to RPA70N relative to aooIn al peptide containing only natural amino acids. The optimized peptides are cell penetrant, able to enter the nucleus, and co-localize with RPA in the nucleus at sites of DNA damage. Such a peptide may serve as a probe molecule to explore both the effects of RPA inhibition on the DNA damage response and the therapeutic potential of RPA inhibition as a cancer target. Citation Format: Alex G. Waterson, Andreas O. Frank, Bhavatarini Vandgamudi, Michael D. Feldkamp, Elaine M. Souza-Fagundes, Jessica W. Luzwick, David Cortez, Edward T. Olejniczak, Olivia W. Rossanese, Walter J. Chazin, Stephen W. Fesik. Optimization of a potent stapled helix peptide that binds to Replication Protein A. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3232. doi:10.1158/1538-7445.AM2014-3232