Proliferating Cell Nuclear Antigen Inhibitor Research Articles

Targeting proliferating cell nuclear antigen enhances ionizing radiation-induced cytotoxicity in prostate cancer cells.

Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, cell growth, and survival. PCNA also enhances androgen receptor (AR) signaling in prostate cancer (PC) cells. We identified a PCNA interaction protein (PIP) box at the N-terminal domain of AR and developed a small peptide PCNA inhibitor R9-AR-PIP containing AR PIP-box. We also identified a series of small molecule PCNA inhibitors (PCNA-Is) that bind directly to PCNA and interrupt PCNA functions. The present study investigated the effects of the PCNA inhibitors on the sensitivity of PC cells to X-ray radiation. The effects of targeting PCNA on radio sensitivity of PC cells were investigated in four lines of castration-resistant PC (CRPC) cells with different AR expression statuses. The cells were treated with the PCNA inhibitors and X-ray radiation alone or in combination. The effects of the treatment on expression of AR target genes, DNA damage response, DNA damage, homologous recombination repair (HRR), and cytotoxicity were evaluated. We found that the androgen response element (ARE) occupancy of the DNA damage response gene PARP1 by AR is significantly attenuated by PCNA-I1S or R9-AR-PIP combined with X-ray radiation, while X-ray radiation alone does not enhance the ARE occupancy. PCNA-I1S or R9-AR-PIP alone significantly inhibits occupancy of the AR-occupied regions (AROR) in PRKDC and XRCC2 genes. R9-AR-PIP and PCNA-I1S inhibit expression of AR-Vs target gene cyclin A2 and show the additive effects with radiation in AR-positive CRPC cells. Targeting PCNA by PCNA-I1S and R9-AR-PIP downregulates expression of DNA damage response genes EXO1, Rad54L, Rad51, and/or PARP1 and shows the additive effects with radiation as compared with their respective controls in AR-positive CRPC LNCaP-AI, 22Rv1, and R1-D567 cells, but not in AR-negative PC-3 cells. R9-AR-PIP and PCNA-I1S elevate the levels of phospho-DNA-PKcs(S2056) and γH2AX, indicating DNA damage in response to radiation in AR-positive cells. The HRR is significantly attenuated by PCNA inhibitors PCNA-I1S, R9-AR-PIP, and T2AA in all four CRPC cells examined, and inhibited by Enzalutamide (Enz) only in 22RV1 cells. The cytotoxicity induced by X-ray radiation in androgen-dependent LNCaP cells is enhanced by Enz and a lower concentration of R9-AR-PIP in the colony formation assay. R9-AR-PIP at higher concentration reduces the colony formation and has an additive effect with X-ray radiation in all AR expressing cells, regardless of AR-FL and AR-Vs, but does not significantly alter the colony formation in AR-negative PC-3 cells. PCNA-I1S attenuates colony formation and has an additive effect with ionizing radiation in all four CRPC cells, regardless of AR expression status. These data provide a strong rationale for the therapy studies using PCNA-I1S or R9-AR-PIP in combination with X-ray radiation against CRPC tumors in preclinical models.

Small molecule targeting of transcription-replication conflict for selective chemotherapy

Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeuticavenue for exploiting this cancer-selective vulnerability.

Proliferating cell nuclear antigen inhibitors block distinct stages of herpes simplex virus infection.

Proliferating cell nuclear antigen (PCNA) forms a homotrimer that encircles replicating DNA and is bound by DNA polymerases to add processivity to cellular DNA synthesis. In addition, PCNA acts as a scaffold to recruit DNA repair and chromatin remodeling proteins to replicating DNA via its interdomain connecting loop (IDCL). Despite encoding a DNA polymerase processivity factor UL42, it was previously found that PCNA associates with herpes simplex virus type 1 (HSV-1) replication forks and is necessary for productive HSV-1 infection. To define the role that PCNA plays during viral DNA replication or a replication-coupled process, we investigated the effects that two mechanistically distinct PCNA inhibitors, PCNA-I1 and T2AA, have on the HSV-1 infectious cycle. PCNA-I1 binds at the interface between PCNA monomers, stabilizes the homotrimer, and may interfere with protein-protein interactions. T2AA inhibits select protein-protein interactions within the PCNA IDCL. Here we demonstrate that PCNA-I1 treatment results in reduced HSV-1 DNA replication, late gene expression, and virus production, while T2AA treatment results in reduced late viral gene expression and infectious virus production. To pinpoint the mechanisms by which PCNA inhibitors affect viral processes and protein recruitment to replicated viral DNA, we performed accelerated native isolation of proteins on nascent DNA (aniPOND). Results indicate that T2AA inhibits recruitment of the viral uracil glycosylase UL2 and transcription regulatory factors to viral DNA, likely leading to a defect in viral base excision repair and the observed defect in late viral gene expression and infectious virus production. In addition, PCNA-I1 treatment results in decreased association of the viral DNA polymerase UL30 and known PCNA-interacting proteins with viral DNA, consistent with the observed block in viral DNA replication and subsequent processes. Together, we conclude that inhibitors of cellular PCNA block recruitment of key viral and cellular factors to viral DNA to inhibit viral DNA synthesis and coupled processes.

A FRET-Based Assay for the Identification of PCNA Inhibitors.

Proliferating cell nuclear antigen (PCNA) is the key regulator of human DNA metabolism. One important interaction partner is p15, involved in DNA replication and repair. Targeting the PCNA-p15 interaction is a promising therapeutic strategy against cancer. Here, a Förster resonance energy transfer (FRET)-based assay for the analysis of the PCNA-p15 interaction was developed. Next to the application as screening tool for the identification and characterization of PCNA-p15 interaction inhibitors, the assay is also suitable for the investigation of mutation-induced changes in their affinity. This is particularly useful for analyzing disease associated PCNA or p15 variants at the molecular level. Recently, the PCNA variant C148S has been associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2). ATLD2 is a neurodegenerative disease based on defects in DNA repair due to an impaired PCNA. Incubation time dependent FRET measurements indicated no effect on PCNAC148S-p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behavior of PCNAC148S was confirmed by intrinsic tryptophan fluorescence, differential scanning fluorimetry (DSF) and asymmetrical flow field-flow fractionation (AF4) measurements. The analysis of the disease associated PCNA variant demonstrated the versatility of the interaction assay as developed.

Identifying Cellular and Viral Factor Recruitment to Herpes Simplex Virus Type 1 Replication Forks

Herpes simplex virus type 1 (HSV‐1) is a ubiquitous pathogen that replicates the 152kbp viral genome within the nucleus of host cells. Replication of the HSV‐1 genome is catalyzed by viral replication machinery consisting of a DNA polymerase (UL30), processivity factor (UL42), helicase‐primase complex, origin binding protein (UL9), and a single stranded DNA binding protein. Despite encoding a viral DNA polymerase processivity factor, we found that host Proliferating Cell Nuclear Antigen (PCNA) interacts with HSV‐1 DNA at replication forks and associates with viral DNA in a replication‐dependent manner. PCNA is a homotrimer that provides processivity to cellular DNA polymerases and selectively recruits DNA damage response and DNA repair factors to cellular replication forks. We therefore hypothesized that PCNA associates at viral replication forks to promote HSV‐1 replication while also tethering cellular DNA repair proteins to replicating viral DNA. To test this, we used two models to identify how associated factors change as a function of PCNA: a known PCNA inhibitor, PCNA‐I1, and a cell line that was engineered to inducibly express a shRNA targeting PCNA. We performed a technique adapted from isolation of proteins on nascent DNA (iPOND) to isolate HSV‐1 DNA from infected cells and identified associated viral and cellular proteins. Cells were infected and replicating viral DNA was selectively labeled with EdC. Labeled DNA was specifically and irreversibly tagged via the covalent attachment of biotin azide via click reaction. Biotin‐tagged DNA was purified on streptavidin‐coated beads and associated proteins were eluted and identified by mass spectrometry. As a complimentary approach, we also performed immunofluorescence (IF) to confirm iPOND data. We found that PCNA is associated with viral DNA despite treatment with PCNA‐I1, an observation that is consistent with IF imaging data. Viral replication proteins UL30, UL42, and UL9 decreased in the presence of PCNA‐I1. DNA damage response proteins such as mismatch repair proteins and RECQL both decreased up to 10‐fold. Of note, however, proteins that make up the MRN complex, a class of proteins that functions in DNA break repair, restart of stalled replication forks, and viral DNA infection, increased in response to PCNA‐I1 treatment. Given this data, PCNA may be involved in tethering viral replication proteins, cellular DNA repair proteins, and/or virion assembly proteins to viral replication forks.

Proliferating cell nuclear antigen directly interacts with androgen receptor and enhances androgen receptor‑mediated signaling.

Androgen receptor (AR) and/or its constitutively active splicing variants (AR‑Vs), such as AR‑V7 and ARv567es, is required for prostate cancer cell growth and survival, and cancer progression. Proliferating cell nuclear antigen (PCNA) is preferentially overexpressed in all cancers and executes its functions through interaction with numerous partner proteins. The aim of the present study was to investigate the potential role of PCNA in the regulation of AR activity. An identical consensus sequence of the PCNA‑interacting protein‑box (PIP‑box) was identified at the N‑terminus of human, mouse and rat AR proteins. It was found that PCNA complexes with the full‑length AR (AR‑FL) and AR‑V7, which can be attenuated by the small molecule PIP‑box inhibitor, T2AA. PCNA also complexes with ARv567es and recombinant AR protein. The PCNA inhibitors, PCNA‑I1S and T2AA, inhibited AR transcriptional activity and the expression of AR target genes in LNCaP‑AI and 22Rv1 cells, but not in AR‑negative PC‑3 cells. The knockdown of PCNA expression reduced dihydrotestosterone‑stimulated AR transcriptional activity and abolished the inhibitory effect of PCNA‑I1S on AR activity. The PCNA inhibitor, PCNA‑I1, exerted additive growth inhibitory effects with androgen deprivation and enzalutamide in cells expressing AR‑FL or AR‑FL/AR‑V7, but not in AR‑negative PC‑3 cells. Finally, R9‑AR‑PIP, a small peptide mimicking AR PIP‑box, was found to bind to GFP‑PCNA at Kd of 2.73µM and inhibit the expression of AR target genes, AR transcriptional activity and the growth of AR‑expressing cells. On the whole, these data strongly suggest that AR is a PCNA partner protein and interacts with PCNA via the PIP‑box and that targeting the PCNA‑AR interaction may represent an innovative and selective therapeutic strategy against prostate cancer, particularly castration‑resistant prostate cancers overexpressing constitutively active AR‑Vs.

Synergistic Lethality between Beta‐lapachone and Proliferating Cell Nuclear Antigen (PCNA) Inhibitor in NQO1‐positive Cancer Cells

β-Lapachone is a classical quinone-containing anti-tumor NQO1 bioactivatable drug which directly kills NQO1 overexpressing cancer cells. However, the clinical applications of β-lapachone are major limited by its high toxicity and modest lethality. To overcome this side effect and expand the therapeutic utility of β-lapachone, we here demonstrate a novel combination therapy of β-lapachone with proliferating cell nuclear antigen (PCNA) inhibitor T2 amino alcohol (T2AA) in various NQO1+ cancer cells. PCNA has a DNA clamp processivity by encircling the DNA double strands to recruit proteins involved in DNA replication and DNA repair. In this study, we found that non-toxic dose of PCNA inhibitor T2AA synergized with sublethal dose of β-lapachone in an NQO1-dependent manner, and combination of T2AA with β-lapachone prevented DNA repair, increased DSB formation and PARP1 hyperactivation, and induced catastrophic energy loss compared to monotherapy. We further defined that T2AA promoted programmed necrosis and S-phase cell cycle arrest in β-lapachone-treated NQO1+ cancer cells.

A cell permeable bimane-constrained PCNA-interacting peptide.

The human sliding clamp protein known as proliferating cell nuclear antigen (PCNA) orchestrates DNA-replication and -repair and as such is an ideal therapeutic target for proliferative diseases, including cancer. Peptides derived from the human p21 protein bind PCNA with high affinity via a 310-helical binding conformation and are known to shut down DNA-replication. Here, we present studies on short analogues of p21 peptides (143–151) conformationally constrained with a covalent linker between i, i + 4 separated cysteine residues at positions 145 and 149 to access peptidomimetics that target PCNA. The resulting macrocycles bind PCNA with KD values ranging from 570 nM to 3.86 μM, with the bimane-constrained peptide 7 proving the most potent. Subsequent X-ray crystallography and computational modelling studies of the macrocyclic peptides bound to PCNA indicated only the high-affinity peptide 7 adopted the classical 310-helical binding conformation. This suggests the 310-helical conformation is critical to high affinity PCNA binding, however NMR secondary shift analysis of peptide 7 revealed this secondary structure was not well-defined in solution. Peptide 7 is cell permeable and localised to the cell cytosol of breast cancer cells (MDA-MB-468), revealed by confocal microscopy showing blue fluorescence of the bimane linker. The inherent fluorescence of the bimane moiety present in peptide 7 allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides. This study identifies a small peptidomimetic that binds PCNA with higher affinity than previous reported p21 macrocycles, and is cell permeable, providing a significant advance toward development of a PCNA inhibitor for therapeutic applications.

Targeting PCNA with Peptide Mimetics for Therapeutic Purposes.

Proliferating cell nuclear antigen (PCNA) is an excellent inhibition target to shut down highly proliferative cells and thereby develop a broad-spectrum cancer therapeutic. It interacts with a wide variety of proteins through a conserved motif referred to as the PCNA-interacting protein (PIP) box. There is large sequence diversity between high-affinity PCNA binding partners, but with conservation of the binding structure-a well-defined 310 -helix. Herein, all current PIP-box peptides crystallised with human PCNA are collated to reveal common trends between binding structure and affinity. Key intra- and intermolecular hydrogen-bonding networks that stabilise the 310 -helix of PIP-box partners are highlighted and related back to the canonical PIP-box motif. High correlation with the canonical PIP-box sequence does not directly afford high affinity. Instead, we summarise key interactions that stabilise the binding structure that leads to enhanced PCNA binding affinity. These interactions also implicate the "non-conserved" residues within the PIP-box that have previously been overlooked. Such insights will allow a more directed approach to develop therapeutic PCNA inhibitors.

Additive effects of a small molecular PCNA inhibitor PCNA-I1Sand DNA damaging agents on growth inhibition and DNA damagein prostate and lung cancer cells.

Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, and cell growth and survival. Previously, we identified a novel class of small molecules that bind directly to PCNA, stabilize PCNA trimer structure, reduce chromatin-associated PCNA, selectively inhibit tumor cell growth, and induce apoptosis. The purpose of this study was to investigate the combinatorial effects of lead compound PCNA-I1S with DNA damaging agents on cell growth, DNA damage, and DNA repair in four lines of human prostate and lung cancer cells. The DNA damage agents used in the study include ionizing radiation source cesium-137 (Cs-137), chemotherapy drug cisplatin (cisPt), ultraviolet-C (UV-C), and oxidative compound H2O2. DNA damage was assessed using immunofluorescent staining of γH2AX and the Comet assay. The homologous recombination repair (HRR) was determined using a plasmid-based HRR reporter assay and the nucleotide excision repair (NER) was indirectly examined by the removal of UV-induced cyclobutane pyrimidine dimers (CPD). We found that PCNA-I1S inhibited cell growth in a dose-dependent manner and significantly enhanced the cell growth inhibition induced by pretreatment with DNA damaging agents Cs-137 irradiation, UV-C, and cisPt. However, the additive growth inhibitory effects were not observed in cells pre-treated with PCNA-I1S, followed by treatment with cisPt. H2O2 enhanced the level of chromatin-bound PCNA in quiescent cells, which was attenuated by PCNA-I1S. DNA damage was induced in cells treated with either PCNA-I1S or cisPt alone and was significantly elevated in cells exposed to the combination of PCNA-I1S and cisPt. Finally, PCNA-I1S attenuated repair of DNA double strand breaks (DSBs) by HRR and the removal of CPD by NER. These data suggest that targeting PCNA with PCNA-I1S may provide a novel approach for enhancing the efficacy of chemotherapy and radiation therapy in treatment of human prostate and lung cancer.

Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy.

The Anticancer Activity of a First-in-class Small-molecule Targeting PCNA.

Proliferating cell nuclear antigen (PCNA) plays an essential role in regulating DNA synthesis and repair and is indispensable to cancer cell growth and survival. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was ubiquitously expressed in a broad range of cancer cells and tumor tissues, but not significantly in nonmalignant cells. We found the L126-Y133 region of caPCNA is structurally altered and more accessible to protein-protein interaction. A cell-permeable peptide harboring the L126-Y133 sequence blocked PCNA interaction in cancer cells and selectively kills cancer cells and xenograft tumors. On the basis of these findings, we sought small molecules targeting this peptide region as potential broad-spectrum anticancer agents. By computer modeling and medicinal chemistry targeting a surface pocket partly delineated by the L126-Y133 region of PCNA, we identified a potent PCNA inhibitor (AOH1160) and characterized its therapeutic properties and potential toxicity. AOH1160 selectively kills many types of cancer cells at below micromolar concentrations without causing significant toxicity to a broad range of nonmalignant cells. Mechanistically, AOH1160 interferes with DNA replication, blocks homologous recombination-mediated DNA repair, and causes cell-cycle arrest. It induces apoptosis in cancer cells and sensitizes them to cisplatin treatment. AOH1160 is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it does not cause significant toxicity at 2.5 times of an effective dose. These results demonstrated the favorable therapeutic properties and the potential of AOH1160 as a broad-spectrum therapeutic agent for cancer treatment.

PCNA dependent cellular activities tolerate dramatic perturbations in PCNA client interactions

Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair, recruiting multiple proteins to their sites of action. We examined the effects of the PCNAS228I mutation that causes PCNA-associated DNA repair disorder (PARD). Cells from individuals affected by PARD are sensitive to the PCNA inhibitors T3 and T2AA, showing that the S228I mutation has consequences for undamaged cells. Analysis of the binding between PCNA and PCNA-interacting proteins (PIPs) shows that the S228I change dramatically impairs the majority of these interactions, including that of Cdt1, DNMT1, PolD3p66 and PolD4p12. In contrast p21 largely retains the ability to bind PCNAS228I. This property is conferred by the p21 PIP box sequence itself, which is both necessary and sufficient for PCNAS228I binding. Ubiquitination of PCNA is unaffected by the S228I change, which indirectly alters the structure of the inter-domain connecting loop. Despite the dramatic in vitro effects of the PARD mutation on PIP-degron binding, there are only minor alterations to the stability of p21 and Cdt1 in cells from affected individuals. Overall our data suggests that reduced affinity of PCNAS228I for specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD.

Target validation and structure-activity analysis of a series of novel PCNA inhibitors.

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.

A site-selective, irreversible inhibitor of the DNA replication auxiliary factor proliferating cell nuclear antigen (PCNA)

Proliferating cell nuclear antigen (PCNA) assumes an indispensable role in supporting cellular DNA replication and repair by organizing numerous protein components of these pathways via a common PCNA-interacting sequence motif called a PIP-box. Given the multifunctional nature of PCNA, the selective inhibition of PIP-box-mediated interactions may represent a new strategy for the chemosensitization of cancer cells to existing DNA-directed therapies; however, promiscuous blockage of these interactions may also be universally deleterious. To address these possibilities, we utilized a chemical strategy to irreversibly block PIP-box-mediated interactions. Initially, we identified and validated PCNA methionine 40 (M40) and histidine 44 (H44) as essential residues for PCNA/PIP-box interactions in general and, more specifically, for efficient PCNA loading onto chromatin within cells. Next, we created a novel small molecule incorporating an electrophilic di-chloro platinum moiety that preferentially alkylated M40 and H44 residues. The compound, designated T2Pt, covalently cross-linked wild-type but not M40A/H44A PCNA, irreversibly inhibited PCNA/PIP-box interactions, and mildly alkylated plasmid DNA in vitro. In cells, T2Pt persistently induced cell cycle arrest, activated ATR-Chk1 signaling and modestly induced DNA strand breaks, features typical of cellular replication stress. Despite sustained activation of the replication stress response by the compound and its modestly genotoxic nature, T2Pt demonstrated little activity in clonogenic survival assays as a single agent, yet sensitized cells to cisplatin. The discovery of T2Pt represents an original effort directed at the development of irreversible PCNA inhibitors and sets the stage for the discovery of analogues more selective for PCNA over other cellular nucleophiles.

Small molecule inhibitors of PCNA/PIP-box interaction suppress translesion DNA synthesis

Proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and DNA damage response. Numerous proteins interact with PCNA through their short sequence called the PIP-box to be promoted to their respective functions. PCNA supports translesion DNA synthesis (TLS) by interacting with TLS polymerases through PIP-box interaction. Previously, we found a novel small molecule inhibitor of the PCNA/PIP-box interaction, T2AA, which inhibits DNA replication in cells. In this study, we created T2AA analogues and characterized them extensively for TLS inhibition. Compounds that inhibited biochemical PCNA/PIP-box interaction at an IC50 <5μM inhibited cellular DNA replication at 10μM as measured by BrdU incorporation. In cells lacking nucleotide-excision repair activity, PCNA inhibitors inhibited reactivation of a reporter plasmid that was globally damaged by cisplatin, suggesting that the inhibitors blocked the TLS that allows replication of the plasmid. PCNA inhibitors increased γH2AX induction and cell viability reduction mediated by cisplatin. Taken together, these findings suggest that inhibitors of PCNA/PIP-box interaction could chemosensitize cells to cisplatin by inhibiting TLS.

Developing peptide-based multivalent antagonists of proliferating cell nuclear antigen and a fluorescence-based PCNA binding assay

Proliferating cell nuclear antigen (PCNA) is a critical player in cell proliferation. It interacts with a myriad of cellular proteins in genomic DNA replication and cell cycle control. This makes PCNA an attractive target for developing antiproliferative therapeutics. Indeed, the binding of a human tumor suppressor protein, p21, to PCNA contributes to its antiproliferative effect in cells. In this work, we report a fluorescence polarization-based binding assay for determining the affinity between the p21 peptide and human PCNA. To improve the potency of the p21-based PCNA antagonist, we exploited the homotrimeric structure of PCNA and developed multivalent peptide-based PCNA antagonists. The di- and trivalent p21-based antagonists bind to PCNA with low nanomolar dissociation constant. Moreover, we show that the multivalent PCNA antagonists inhibited PCNA-dependent DNA synthesis in a human cell extract with improved avidity when compared with the monovalent p21 peptide. The fluorescence polarization assay holds promise for the discovery of potent small-molecule PCNA inhibitors given its ready adaptability to a high-throughput screening format.

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 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.

Small-Molecule Targeting of Proliferating Cell Nuclear Antigen Chromatin Association Inhibits Tumor Cell Growth

Proliferating cell nuclear antigen (PCNA), a potential anticancer target, forms a homotrimer and is required for DNA replication and numerous other cellular processes. The purpose of this study was to identify novel small molecules that modulate PCNA activity to affect tumor cell proliferation. An in silico screen of a compound library against a crystal structure of PCNA and a subsequent structural similarity search of the ZINC chemical database were carried out to derive relevant docking partners. Nine compounds, termed PCNA inhibitors (PCNA-Is), were selected for further characterization. PCNA-I1 selectively bound to PCNA trimers with a dissociation constant (K(d)) of ~0.2 to 0.4 μM. PCNA-Is promoted the formation of SDS-refractory PCNA trimers. PCNA-I1 dose- and time-dependently reduced the chromatin-associated PCNA in cells. Consistent with its effects on PCNA trimer stabilization, PCNA-I1 inhibited the growth of tumor cells of various tissue types with an IC(50) of ~0.2 μM, whereas it affected the growth of nontransformed cells at significantly higher concentrations (IC(50), ~1.6 μM). Moreover, uptake of BrdU was dose-dependently reduced in cells treated with PCNA-I1. Mechanistically the PCNA-Is mimicked the effect of PCNA knockdown by siRNA, inducing cancer cell arrest at both the S and G(2)/M phases. Thus, we have identified a class of compounds that can directly bind to PCNA, stabilize PCNA trimers, reduce PCNA association with chromatin, and inhibit tumor cell growth by inducing a cell cycle arrest. They are valuable tools in studying PCNA function and may be useful for future PCNA-targeted cancer therapy.

Differential Requirement for Proliferating Cell Nuclear Antigen in 5′ and 3′ Nick-directed Excision in Human Mismatch Repair

Proliferating cell nuclear antigen (PCNA) is involved in mammalian mismatch repair at a step prior to or at mismatch excision, but the molecular mechanism of this process is not fully understood. To examine the role of PCNA in mismatch-provoked and nick-directed excision, orientation-specific mismatch removal of heteroduplexes with a pre-existing nick was monitored in human nuclear extracts supplemented with the PCNA inhibitor protein p21. We show here that, whereas 3' nick-directed mismatch excision was completely inhibited by low concentrations of p21 or a p21 C-terminal fusion protein, 5' nick-directed excision was only partially blocked under the same conditions. No further reduction of the 5' excision was detected when a much higher concentration of p21 C-terminal protein was used. These results suggest the following. (i) There is a differential requirement for PCNA in 3' and 5' nick-directed excision; and (ii) 5' nick-directed excision is conducted by a manner either dependent on or independent of PCNA. Our in vitro reconstitution experiments indeed identified a 5' nick-directed excision pathway that is dependent on PCNA, hMutSalpha, and a partially purified fraction from a HeLa nuclear extract.