Discovery of New RPA-70 Inhibitors With the Potentiation of Acting as Chemosenitizing Agents

Objectives: Determination of compounds that induce synthetic-lethality through inhibition of replication protein A (RPA). RPA is crucial for DNA replication, repair, and damage-signaling and possesses three subunits, (RPA-1, RPA-2, and RPA-3) composed of six smaller ssDNA-binding domains (DBD A-F). Found on the largest subunit, RPA-1, DBD-F consists of a binding cleft that readily binds both ssDNA and proteins involved in DNA metabolism. While RPA binds to ssDNA through DBDs A-E, DBD-F binds to proteins essential for DNA repair. Through pharmacological manipulation and further testing, compounds that inhibit the DBD-F are being investigated as a potential cancer therapy through the inhibition of DNA repair. The possibility of increasing the efficacy of conventional cancer treatments while decreasing toxic effects is explored.
Methods: For the high-throughput screen (HTS), pre-loaded 384-well plates, consisting of buffer and compound were provided. Each well was combined with a dilution of a quenched fluorescent dsDNA probe and RPA and then allowed to incubate at room temperature for five minutes before being loaded and read on an infrared scanner. In this assay, inhibition of DNA unwinding, and subsequent unquenching, is indicative of DBD-F inhibition. Then, as a counterscreen, compounds were run in an Electrophoretic Mobility Shift Assay (EMSA) to determine if the compound inhibited other DBDs invovled in ssDNA binding. Ultimately, compounds that inhibited the negatively charged cleft on the F-domain, while still allowing for ssDNA binding were recorded for further investigation.
Results: Out of a possible 5000 compounds tested, eight compounds were found to have strong DBD-F inhibition while not inhibiting other DBDs on RPA. Upon further investigation through Chembridge these eight compounds were determined to possess characteristics of pharmaceutical drugs.
Conclusions: Extensive research on these eight compounds must be completed before conclusive results can be drawn. However, it can be determined that these compounds are potential chemosenitizing agents.