Abstract 3340: Stapled helix peptides as probes to evaluate targeted disruption of protein-protein interactions mediated by RPA70N.
Authors: Vangamudi Bhavatarini , Frank Andreas O. , Souza-Fagundes Elaine M. , Feldkamp Michael D. , Olejniczak Edward T. , Rossanese Olivia W. , Fesik Stephen W. 
Published: August 2014

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 RPA 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 are an emerging technology for the inhibition of protein-protein interactions. Incorporation of a hydrocarbon “staple” has the potential to increase the potency, stability, and cell permeability of peptides. Here, we report the development 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.

An initial alanine scan of the native ATRIP-derived sequence identified residues critical for peptide binding to RPA70N. In addition, the scan revealed residue positions that were dispensable and therefore suitable as sites for incorporation of a staple. Introduction of a conserved WFA motif derived from analysis of the p53-binding sequence produced a 10-fold gain in potency over the native ATRIP peptide. To facilitate entry into cells, negatively charged residues were replaced by alanines or by neutral, polar residues. In most instances, residues that improved the net charge had a deleterious effect on binding affinity. The resulting peptide, chosen for stapling, represented a balance between net charge and potency and was intended to offer the best chance of cell penetration while still maintaining affinity. Based on the alanine scan data, two positions were chosen for incorporation of a hydrocarbon staple; however, only one of these stapled peptides maintained binding affinity for RPA70N. The optimized peptide was cell penetrant, able to enter the nucleus, and co-localized with RPA in the nucleus at sites of DNA damage. In this study, we further examine the functional consequences of RPA70N disruption by ATRIP-derived hydrocarbon stapled peptides and discuss the use of them as tools to probe the therapeutic relevance of RPA inhibition in breast and other cancers.

Citation Format: Bhavatarini Vangamudi, Andreas O. Frank, Elaine M. Souza-Fagundes, Michael D. Feldkamp, Edward T. Olejniczak, Olivia W. Rossanese, Stephen W. Fesik. Stapled helix peptides as probes to evaluate targeted disruption of protein-protein interactions mediated by RPA70N. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3340. doi:10.1158/1538-7445.AM2013-3340