Due to the high rate of DNA damage within the cell cycle, DNA damage response and repair pathways are critical for the survival of eukaryotic cells. Beyond the survival of healthy cells, DNA repair pathways also play a role in the survival of cancer cells. The inhibition of these repair proteins represent a potential strategy for cancer therapy. The inhibition of these repair proteins represent a potential strategy for cancer therapy. Identifying and validating different targets within DNA damage response pathways is a strategy for attaining novel cancer targets from a validated paradigm. Replication protein A (RPA) was identified as a potential novel target for triple negative breast cancer (TNBC) from a siRNA screen of 20 TNBC cell lines performed by the Cortez group at Vanderbilt. RPA is a ssDNA binding protein responsible for recruiting and initiating multiple DNA damage response proteins such as Rad9, MRE11, and ATRIP and as such serves as both a G1/S and G2/M cell cycle checkpoint. Our hypothesis is that the inhibition of the protein-protein binding interface of RPA70N should allow for the blockage DNA damage response pathways and thus induce apoptosis in cancer cells. Due to the inherent difficulties with inhibiting protein-protein interactions, a fragment-based discovery approach will be undertaken in order to discover a selective inhibitor of the protein-protein binding interface of RPA70N. We will be using the technique of "SAR by NMR" to discover the lead molecule of appropriate potency. A 15,000 member fragment library has been screened using 1H/15N HSQC. We have identified 150 fragment hits from our library and currently have eleven co-crystal structures of molecules bound to RPA70N. Small libraries of the fragment hits will be generated and tested via 1H/15N HSQC to establish the SAR of the fragment molecules. Once the fragments have been optimized, we will link the two fragments together using a small subset of flexible linkers to generate a lead molecule. The lead molecule will be further optimized using structural information to fill any and all unoccupied binding nooks and crannies on RPA70N. Simultaneously, the pharmaceutical properties of the lead molecule will be taken into consideration and optimized. The lead molecule of sufficient potency and pharmaceutical properties will be used to validate our hypothesis that the inhibition of protein-protein interactions of RPA70N will lead to cancer cell death.
Replication protein A (RPA), a ssDNA binding protein responsible for recruiting and initiating DNA damage response proteins as a cell cycle checkpoint, was recently identified as a potential target for triple negative breast cancer. Fragment-based drug discovery, a technique known for generating novel chemical matter and inhibiting protein-protein interactions, will be used to discover selective inhibitors of the RPA70 protein-protein binding interface. These molecules will be optimized for binding to RPA70N as well as pharmaceutical properties, in order for them to be used as chemical probes to validate the inhibition of the protein-protein interactions of RPA as a potential cancer target.
|Feldkamp, Michael D; Frank, Andreas O; Kennedy, J Phillip et al. (2013) Surface reengineering of RPA70N enables cocrystallization with an inhibitor of the replication protein A interaction motif of ATR interacting protein. Biochemistry 52:6515-24|