Abstract
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 IC(50) 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(Ser(139))histone H2AX induction and cell growth inhibition was observed.
Highlights
proliferating cell nuclear antigen (PCNA) is a multifunctional component of DNA replication and repair machinery
T3 Binds to PCNA Cavity That Interacts with PIP-box Sequences—We have established a high-throughput screening protocol to find agents that inhibit biochemical PCNA/PIP-box interactions. This method uses conventional fluorescence polarization (FP) for screening chemical libraries by competing the binding of recombinant PCNA protein and fluorescently tagged PL-peptide that possesses a PIP-box sequence, which was previously optimized for high PCNA affinity (IC50 of ϳ100 nM in self-competition; data not shown) [11]
After eliminating nonspecific compounds by establishing dose-response curves for the hit compounds with at least two independent experiments, we have ensured that T3 (Fig. 1A) is a true inhibitor of PCNA/PL-peptide binding at IC50 of ϳ3 M (Fig. 1C)
Summary
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. 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. Often these agents are not therapeutically efficacious in diverse cancers, presumably because tumors can be supported by multiple mechanisms (e.g. not “addictive” to the pathway targeted) This leads to failure of proof-of-concept validation in the cancer drug discovery programs. We have created a small molecular inhibitor of the PCNA/PIP-box interaction and investigated its pharmacological effects in cells from a chemotherapeutic viewpoint
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