Abstract PR02: Small molecules that specifically inhibit the D-loop activity of RAD51

RAD51 plays a central role in homologous recombination (HR), which maintains genome integrity. RAD51 is commonly overexpressed in cancer cells relative to normal tissue and is considered a therapeutic target in oncology. One potential challenge for targeting RAD51 pharmacologically is that it mediates functions in both double-strand DNA break (DSB) repair and stabilization of stalled replication forks. In order to distinguish compound-mediated effects on these RAD51 functions, we developed a novel class of RAD51 inhibitors. In contrast to many previously reported RAD51 inhibitors, we sought to develop compounds that do not inhibit RAD519s ability to bind single-stranded DNA (ssDNA). Instead, these compounds prevent RAD51-ssDNA nucleoprotein filaments from invading into homologous double-stranded DNA templates and forming D-loops. Our initial lead compound RI(dl)-1 (an abbreviation for RAD51 inhibitor of D-loop formation #1) is capable of blocking RAD51-mediated D-loop formation in biochemical assays, using concentrations that do not prevent RAD51 binding to ssDNA. An analog of this compound, termed RI(dl)-2, provides even better inhibition of RAD519s D-loop activity in biochemical systems (IC50 15.8 µM). RI(dl)-2 reduces HR activity in cells in dose ranges that do not stimulate single strand annealing (SSA) activity, which distinguishes it more generalized RAD51 inhibitors. RI(dl)-2 also sensitizes several cancer cell lines to radiation-induced death. We now present the results of more extensive structure activity relationship (SAR) optimizations aimed at further improving the potency and selectivity of this class of compounds. Several of these compounds are capable of delaying the timely resolution of radiation-induced RAD51 foci, even though initial RAD51 foci are apparently normal at earlier time points. Similar focus kinetics are observed for gamma-H2AX in identically treated cells, supporting the interpretation that these compounds permit RAD51 assembly at DSBs but prevent the completion of DSB repair. Unlike RI(dl)-2, some these newer compounds sensitize cancer cells to mitomycin C. These specialized RAD51 inhibitory compounds are attractive candidates for potential use as radiation or chemotherapy sensitizers, since they may exert fewer toxic risks compared to more generalized inhibitors of the HR repair machinery. They may also serve to help distinguish the functions of RAD51 DSB repair and replication stress tolerance, which may enable better understanding of RAD519s roles in maintaining genome stability. This abstract is also being presented as Poster A02. Citation Format: Brian Budke, Wei Lv, Werner Tueckmantel, Alan Kozikowski, Philip Connell. Small molecules that specifically inhibit the D-loop activity of RAD51 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR02.