Development of a high-throughput screen for inhibitors of replication protein A and its role in nucleotide excision repair.
The heterotrimeric protein, replication protein A (RPA), is essential for DNA repair and replication. RPA is a viable target in the treatment of cancer as many chemotherapeutic agents act by blocking DNA replication. Furthermore, inhibition of RPA could prove useful in treating cancers that have acquired resistance to DNA damaging agents through enhanced DNA repair mechanisms as has been observed with certain platinum-resistant carcinomas. In an effort to identify inhibitors of RPA, we employed a novel fluorescent reporter and established a homogeneous high-throughput screening assay to measure RPA's DNA binding activity. Using this assay, we have screened a collection of small molecules and determined the effect they have on the RPA-DNA interaction. Of the 2000 compounds screened, 79 scored positive for inhibition of RPA binding activity. Secondary screenings were performed using an electrophoretic mobility shift assay; of the 79 compounds, 9 scored positive and were further characterized in titration experiments to determine the most potent inhibitor, resulting in several compounds showing an IC50 in the low micromolar range. Fluorescence polarization analyses were also performed to determine the mechanism of inhibition for each compound. Validation of the inhibitory activity of selected compounds was verified using in vitro nucleotide excision repair (NER) catalyzed excision of a single cisplatin lesion in a duplex DNA. The identification and use of RPA inhibitors may aid in inhibiting NER activity that could potentially circumvent resistance to certain chemotherapeutic agents as well as be useful in the characterization of RPA and its interaction with DNA.
Andrews
Brooke J
BJ
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA.
Turchi
John J
JJ
D000970:Antineoplastic Agents / Q000494:pharmacology; D001667:Binding, Competitive; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D019008:Drug Resistance, Neoplasm; D024202:Electrophoretic Mobility Shift Assay; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D011485:Protein Binding / Q000187:drug effects; D051716:Replication Protein A; D015203:Reproducibility of Results
D000970:Antineoplastic Agents; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; C497167:RPA1 protein, human; D051716:Replication Protein A