Replication Protein A (RPA) is a heterotrimeric protein that binds to and protects ssDNA and plays an integral role in initiating the cellular response to DNA damage. This response is mediated via protein-protein interactions between a basic cleft on the RPA70N subunit and a number of protein partners, including ATRIP, Rad9, Mre11, and p53. RNAi against RPA has shown an expected toxicity against cancer cell lines, possibly due to abrogation of the ssDNA binding function of RPA. Specific disruption of the protein-protein interactions between the RPA07N subunit and its binding partners has the potential to produce a more selective cytotoxic response in cancer cells. To more accurately dissect the therapeutic relevance of disrupting only the protein-protein interaction functions of RPA, we sought to discover potent small molecule probes that bind to the basic cleft of RPA70N.

Inhibition of protein-protein interactions is considered a difficult task. An NMR-based fragment screen has identified more than 130 fragment molecules that bind to the RPA70N protein-protein interaction cleft with affinities that range from 500 μM to 2 mM and corresponding ligand efficiencies from 0.18 to 0.30. Selected fragments, representing several distinct chemotypes, have been optimized for binding to the protein. Using X-ray crystallography, the binding modes of these fragments have been defined. Fragments were found to bind primarily to two main sites within the basic cleft of RPA70N. Additional structure-guided optimizations have been carried out and ternary co-crystal structures have been generated to guide fragment linking strategies. Together, these activities have led to the creation of multiple lead series of inhibitors of the RPA:ATRIP interaction, with binding affinities improved by several fold over the initial fragments. The SAR and biological activities of the fragments and lead compounds will be discussed.

Citation Format: Alex G. Waterson, James D. Patrone, J. Phillip Kennedy, Nicholas F. Pelz, Andreas O. Frank, Bhavatarini Vandgamudi, Michael D. Feldkamp, Elaine M. Souza-Fagundes, Olivia W. Rossanese, Walter J. Chazin, Stephen W. Fesik. Fragment-based discovery of inhibitors of replication protein A protein-protein interactions. [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 2473. doi:10.1158/1538-7445.AM2013-2473