Fragment-based discovery: Small molecule inhibitors of XPA-RPA

Project Summary Nucleotide excision repair (NER) is the primary pathway used to repair bulky DNA adducts, which are caused by diverse exposures ranging from UV light to certain chemotherapeutic agents. Although NER is necessary for protecting human cells from DNA damage, the pathway can significantly reduce the efficacy of cancer therapeutics that damage DNA, such as cisplatin. Cisplatin is a front-line treatment for a variety of cancer types; however, many patients develop resistance to the drug. Our long-range goal is to develop strategies to improve treatment response. This proposal investigates the hypothesis that small molecules inhibiting the interaction between two critical NER proteins, Xeroderma Pigmentosum Complementation Group A (XPA) and Replication Protein A (RPA), will lead to reduced NER capacity and increased Pt-agent sensitivity. The Chazin lab has previously (i) mapped the interaction between XPA and RPA, (ii) determined that XPA mutations known to disrupt binding with RPA decrease NER activity, and (iii) shown that disruption of the NER pathway seems to correlate with increased Pt-agent response. The objectives of this proposal are to generate small molecule inhibitors of the XPA-RPA interaction (Aim 1) and test their ability to diminish NER efficacy and sensitize cells to Pt-agents. Aim 1 will utilize a fragment-based discovery approach, screening a highly curated library of small molecular fragments. NMR will be used to identify fragment hits that bind within the XPA-RPA interface. These will be elaborated and optimized, and hits occupying different sites in the interaction interface will be linked to generate higher affinity compounds. Fragments and linked compounds will undergo multiple rounds of optimization so that the most promising inhibitor candidates will be developed. Aim 2 will determine the effect of candidate inhibitors on physical interaction of XPA and RPA, NER activity and Pt-agent sensitivity. The mode of action and binding affinity of the inhibitors will be characterized with techniques including NMR and fluorescence-based competition assays to confirm their ability to inhibit the interaction. Select inhibitors will then be tested in a variety of cell- based assays to determine if they diminish NER efficiency and increase sensitivity to Pt-agents. These aims will generate valuable tool compounds that provide detailed insight into the correlation between NER activity and response to Pt-based agents and serve as a foundation for testing the potential therapeutic value of inhibiting NER to improve the response to Pt-based anticancer therapies.