Identification of Small Molecule Proliferating Cell Nuclear Antigen (PCNA) Inhibitor That Disrupts Interactions with PIP-box Proteins and Inhibits DNA Replication

We have discovered that 3,3′,5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC50 of ∼1 μm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser139)histone H2AX induction and cell growth inhibition was observed. Background: PCNA is a multifunctional component of DNA replication and repair machinery. Results: A novel small molecule inhibitor of the PCNA protein-protein interaction inhibited DNA replication, induced DNA replication stress, and increased cisplatin-mediated DNA damage response in cells. Conclusion: The biochemical PCNA inhibitor can inhibit PCNA functions essential for cells. Significance: Inhibition of the PCNA protein-protein interaction can be a new strategy to sensitize cancer cells to chemotherapy.