Validating the disruption of proliferating cell nuclear antigen interactions in the development of targeted cancer therapeutics

Abstract

Human DNA replication and repair is a highly coordinated process involving the specifically timed actions of numerous proteins and enzymes. Many of these proteins require interaction with proliferating cell nuclear antigen (PCNA) for activation within the process. The interdomain connector loop (IDCL) of PCNA provides a docking site for many of those proteins, suggesting that this region is critically important in the regulation of cellular function. Previous work in this laboratory has demonstrated that a peptide mimicking a specific region of the IDCL (caPeptide) has the ability to disrupt key protein-protein interactions between PCNA and its binding partners, thereby inhibiting DNA replication within the cells. In this study, we confirm the ability of the caPeptide to disrupt DNA replication function using both intact cell and in vitro DNA replication assays. Further, we were able to demonstrate that treatment with caPeptide results in a decrease of polymerase d activity that correlates with the observed decrease in DNA replication. We have also successfully developed a surface plasmon resonance (SPR) assay to validate the disruption of the PCNA-pol d interaction with caPeptide.Abbreviations: PCNA, proliferating cell nuclear antigen; IDCL, interdomain connector loop; caPeptide, cancerassociated peptide; RF-C, replicating factor C; FEN-1, flap endonuclease 1; RPA, replication protein A; PIP-box, PCNAinteracting protein box; pI, isoelectric point; caPCNA, cancer-associated PCNA; DNA, deoxyribonucleic acid; RNA, ribonucleic acid; SV40, Simian virus 40; T-ag, T antigen; EC 50, , 50% effective dose; SPR, surface plasmon resonance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; pol d, polymerase d; pol a, polymerase a; scrPeptide, scrambled peptide; K D, , equilibrium dissociation constants; POLD3, partial recombinant pol d of p68 subunit; k a, , association constant; k d, , dissociation constant; DMEM, Dulbecco's Modified Eagle Media (DMEM); PBS, phosphate buffered saline; DMSO, dimethyl sulfoxide; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate; EDTA, ethylenediaminetetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; RNA, ribonucleic acid; TBE, 50 mM Tris/ 90 mM boric acid/ 2 mM EDTA; PCR, polymerase chain reaction; HBS-EP, HEPES buffered saline with EDTA and P20

Molecular Targeting of Cancer-Associated PCNA Interactions in Pancreatic Ductal Adenocarcinoma Using a Cell-Penetrating Peptide

“…In this study, we show that R9-caPeptide inhibits replication fork progression, causes DNA damage, and ultimately leads to apoptosis of pancreatic cancer cells. [21][22][23] Importantly, R9-caPeptide is minimally toxic to normal or non-cancerous immortalized cells as reported in our previous work. 19,21 The observed inhibition of replication progression is likely mediated through disruption of PCNA-FEN1 and PCNA-LIG1 interactions that are important in the maturation of Okazaki fragments, compromising replication fork progression, as shown previously.…”

“…We showed that caPeptide inhibited in vitro DNA replication due to its ability to disrupt specific PCNA interactions with various DNA replication components, including Pol d and FEN1. 21,22 Furthermore, when attached to an R9 peptide (nine arginines) for cellular delivery, the R9-caPeptide exhibited cytotoxicity in breast and neuroblastoma cancer cell lines. It was also shown that R9-caPeptide is non-toxic to non-malignant cell lines, such as human peripheral blood mononuclear cells, human neural crest stem cells, and human mammary epithelial cells.…”

Therapeutic Targeting of DNA Replication Stress in Cancer

“…A cell permeable peptide (R9-caPep) containing the L126-Y133 sequence of PCNA selectively blocks PCNA interactions in cancer cells and interferes with DNA synthesis and HR-mediated DSB repair, resulting in S-phase arrest, an accumulation of DNA damage, and an enhanced sensitivity to cisplatin [9]. R9-caPep also selectively kills cancer cells with much less toxicity to human peripheral blood mononuclear cells or neural crest stem cells and suppresses cell growth in a mouse xenograft model without causing any discernable toxicity to the animals [9,[185][186][187]. These findings demonstrate that targeting protein-protein interactions involving the L126-Y133 region of PCNA may prove to be an effective approach to treating cancers with reduced side effects.…”

Novel Peptide Therapeutic Approaches for Cancer Treatment

“…R9-caPeptide was found to disrupt interaction of PCNA with its binding partners. Surface plasmon resonance studies showed disruption of PCNA with a peptide from FEN1 and the p66 subunit of DNA polymerase δ (POLD3) [ 105 , 106 ]. Additionally, caPeptide was found to interact with POLD3 [ 107 ].…”