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

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.

Oxidative Stress, DNA, Cell Cycle/Cell Cycle Associated Proteins and Multidrug Resistance Proteins: Targets of Human Amniotic Membrane in Hepatocellular Carcinoma

“…Since hAMPE induced G2/M phase arrest in HuH7 cell lines, it was likely that increased P21 inhibited PCNA. Since PCNA acts as a scaffold protein that organizes several components for DNA replication or cell cycle progression, PCNA inhibition could probably be responsible for several experimental results presented by our team in this and other papers [13,42]. On the other hand, since hAMPE induced a high DNA damage in HepG2 cell line, and since G2/M checkpoint is essential for maintaining genome stability, the HepG2 cell cycle arrest in this phase could be easily explained [43].…”

Deviating the level of proliferating cell nuclear antigen inTrypanosoma bruceielicits distinct mechanisms for inhibiting proliferation and cell cycle progression

“…This has fundamentally restricted the types of studies for chemotherapeutic interventions of PCNA to human proliferative diseases such as cancer, arthritis and nephritis. 52,[56][57][58][59] The results of this study validate TbPCNA as a viable target for therapeutic intervention against African trypanosomiasis and broaden the categories of where PCNA therapeutics may be beneficial to infectious disease research. Future studies will elucidate mechanisms of TbPCNA regulation in T. brucei.…”

“…Information gained from such studies can also reveal how deregulating TbPCNA triggers G2/M arrest. This information in combination with that obtained from several novel classes of PCNA inhibitors 52,59 may lead to the development of innovative therapies that target components of the T. brucei DNA replication machinery.…”

Structure of p15PAF–PCNA complex and implications for clamp sliding during DNA replication and repair

“…Our proposed model for p15-modulated PCNA sliding along the DNA could in future be validated by single-molecule experiments 40 . The PIP-box/PCNA interaction is a promising target for cancer therapeutics 43,44 . Yet, PCNA directs fundamental DNAtemplated processes, and targeting its common interaction site for so many different PIP-box proteins might cause unspecific cytotoxicity.…”