Abstract
Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair. Tumor cells express high levels of PCNA, identifying it as a potentially ideal target for cancer therapy. Previously, we identified nine compounds termed PCNA inhibitors (PCNA-Is) that bind directly to PCNA, stabilize PCNA trimer structure, reduce chromatin-associated PCNA, and selectively inhibit tumor cell growth. Of these compounds, PCNA-I1 was most potent. The purpose of this study is to further establish targeting of PCNA by PCNA-I1 and to identify PCNA-I1 analogs with superior potencies. We found that PCNA-I1 does not affect the level of chromatin-associated PCNA harboring point mutations at the predicted binding site of PCNA-I1. Forty-six PCNA-I1 analogs with structures of 1-hydrazonomethyl-2-hydroxy (scaffold A), 2-hydrazonomethyl-1-hydroxy (scaffold B), 2-hydrazonomethyl-3-hydroxy (scaffold C), and 4-pyridyl hydrazine (scaffold D) were analyzed for their effects on cell growth in four tumor cell lines and PCNA trimer stabilization. Compounds in scaffold group A and group B showed the highest trimer stabilization and the most potent cell growth inhibitory activities with a significant potency advantage observed in the Z isomers of scaffold A. The absence of trimer stabilization and growth inhibitory effects in compounds of scaffold group D confirms the essentiality of the hydroxynaphthyl substructure. Compounds structure–activity relationship (SAR)-6 and SAR-24 were analyzed for their effects on and found to reduce chromatin-associated PCNA in tumor cells. This study led to the identification of SAR-24, a compound with superior potencies and potentially improved solubility, which will be used for future development of PCNA-targeting cancer therapies.
Highlights
Proliferating cell nuclear antigen (PCNA) is an evolutionally well-conserved nononcogenic protein ubiquitously expressed in all types of cells
To determine whether the predicted binding of PCNA-I1 to these amino acid residues is necessary for the effects of PCNA-I1 on chromatin association, we assessed the affects of PCNA-I1 on chromatin association of mutant PCNA
Both wild-type and mutated green fluorescent protein (GFP)-PCNAs can localize to nucleus (Fig. 1B) and associate to chromatin, which is revealed by their presence in the NP-40 extraction-resistant fraction (NP-R, GFP-PCNA in the upper panel of Figure 1C)
Summary
Proliferating cell nuclear antigen (PCNA) is an evolutionally well-conserved nononcogenic protein ubiquitously expressed in all types of cells. PCNA plays crucial roles in numerous cellular processes, such as DNA replication and repair, cell survival, cell cycle control, and chromatin assembly (Kelman and Hurwitz 1998; Moldovan et al 2007; Naryzhny 2008; Stoimenov and Helleday 2009) It executes these crucial roles through interaction with over 400 protein partners, including DNA polymerase d and e for DNA replication, DNMT1, HDAC1, and p300 for chromatin assembly and gene regulation, DNA mismatch repair protein Msh and Msh for DNA repair, p21, p15, cyclin D1, and CDK2 for cell cycle control, and ESCO1 and ESCO2 for sister-chromatid cohesion (Maga and Hubscher 2003; Stoimenov and Helleday 2009). The critical importance of PCNA for cell growth and survival is underscored by the finding that a homozygous deletion of PCNA is embryonically lethal in mice (Roa et al 2008)
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