poly(ADP-ribose) Polymerase Family Members Research Articles

Poly(ADP-Ribose) Polymerase (PARP) Inhibitors for Cancer Therapy: Advances, Challenges, and Future Directions

Poly(ADP-ribose) polymerases (PARPs) are crucial nuclear proteins that play important roles in various cellular processes, including DNA repair, gene transcription, and cell death. Among the 17 identified PARP family members, PARP1 is the most abundant enzyme, with approximately 1–2 million molecules per cell, acting primarily as a DNA damage sensor. It has become a promising biological target for anticancer drug studies. Enhanced PARP expression is present in several types of tumors, such as melanomas, lung cancers, and breast tumors, correlating with low survival outcomes and resistance to treatment. PARP inhibitors, especially newly developed third-generation inhibitors currently undergoing Phase II clinical trials, have shown efficacy as anticancer agents both as single drugs and as sensitizers for chemo- and radiotherapy. This review explores the properties, characteristics, and challenges of PARP inhibitors, discussing their development from first-generation to third-generation compounds, more sustainable synthesis methods for discovery of new anti-cancer agents, their mechanisms of therapeutic action, and their potential for targeting additional biological targets beyond the catalytic active site of PARP proteins. Perspectives on green chemistry methods in the synthesis of new anticancer agents are also discussed.

Abstract 4698: Measuring cellular PARylation to gain insight in PARP/PARG-targeted drug discovery

Abstract Inhibitors of poly (ADP-ribose) polymerases (PARPs) have revolutionized cancer therapy by highlighting the potential of synthetic lethal drugs to target the DNA damage response (DDR) network. More recently, the development of PARG inhibitors was spurred by the discovery that inhibiting PARG (poly (ADP-ribose) glycohydrolase) results in the accumulation of poly ADP-ribose on DDR proteins and induces cell death. Adding PARG or PARP inhibitors to a cell of interest and quantifying the resulting levels of PARylation can provide valuable insight on compound membrane permeability and target engagement in a cellular context. Nevertheless, assessing PARylation (poly-ADP-ribosylation in which multiple ribose units are added in linear or branched forms) in cells is difficult compared to biochemical assays, impeding the progress of candidate drug development. A sandwich ELISA-based assay was developed to analyze the total protein PARylation present in cellular extracts. Experimental protocols were optimized to discern differences in cellular PARylation levels resulting from activating the DDR and from exposure to PARP inhibitors and/or PARG inhibitors. It is worth noting that our assay specifically detects PARylation, measuring the effects associated with PARP family members 1 to 5, as other family members primarily catalyze MARylation (mono-ADP ribosylation in which a single ADP-ribose unit is added). Titration of known PARP inhibitors and PARG inhibitors was performed to validate the assay. 1) PARG inhibitor PDD00017273, used in combination with DNA damaging agent H2O2, increased the level of cellular PARylation, exhibiting a similar IC50 value across the four cell lines tested. This is consistent with the notion that PARG is the major de-PARylating enzyme in most cells. The potency of other known PARG inhibitors PDD00017272 and PDD00017238 ranked the compounds as expected from published results (PDD00017272< PDD00017273< PDD00017238). 2) Titration of the three PARP1/2 inhibitors Talazoparib, Olaparib and AZD5305, in combination with a DNA damage agent and PDD00017273 inhibitor, shows a dose-dependent reduction in cellular PARylation in HEK293 cells, as expected since PARP1 is activated in response to DNA damage and this activation is blocked by the inhibitors (IC50 values, Olaparib = 0.013 µM, AZD5305 = 0.0024 µM, Talazoparib = 0.0016 µM). These IC50 are consistent with published values and underscore the usefulness of the assay in measuring dose response effects, allowing accurate determination of compound IC50 in living cells. In summary, our optimized experimental cell treatment protocols and ELISA-based assay allow quantification of total cellular PARylation and accurately rank candidate drugs targeting PAR erasers or PAR writers in a high-throughput format, using living cells. Citation Format: Caila Pilo, Mohammad Awad, Veronique T. Baron, Fernando Martins, Jonathan Mikolosko, Steven Plouffe, Zhihua Tao, Katarzyna Zientara-Rytter, Henry Zhu, Pavel Shashkin. Measuring cellular PARylation to gain insight in PARP/PARG-targeted drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4698.

Abstract 7114: Exploring the landscape of PARP and PARG inhibitor selectivity in live cells using NanoBRET™ target engagement assays

Abstract The 17-member poly(ADP-ribose) polymerase (PARP) family of proteins mediate a number of cellular processes including gene transcription, cell-division and DNA-repair. PARPs catalyze the addition of poly- or mono- ADP-ribose chains on target proteins, modifying their function and/or recruitment in various pathways. Poly(ADP-ribose) Glycohydrolase (PARG) catabolizes PAR units and facilitates the recycling of PARP and other modified effector proteins. A number of PARPS are implicated in human disease, and PARP inhibitors (PARPi’s) have seen major clinical success, however, many PARPs (especially mono PARPs) are understudied and chemical tools for understanding cellular PARPi selectivity and potency across the entire family are lacking. Similarly, several PARG inhibitors have been developed to date, but tools for directly assessing target engagement in a cellular context are sorely needed. Here we describe the development and application of target engagement assays for characterizing PARP and PARG inhibitor selectivity and affinity in live cells. This method is based on the NanoBRET™ assay platform, which is a proximity-based probe displacement assay that operates in live cells. Using NanoBRET™ technology, we have developed a single probe that covers 12 out of 17 PARP family members. We explored the family-wide selectivity of a diverse panel of clinical and preclinical PARP inhibitors in live cells, representing the first family-wide, cell-based method for profiling PARP selectivity. We also describe the development of a second probe that reports on PARG target engagement. Target engagement of PARG was observed for three out of five tested inhibitors, suggesting a potential indirect mode of action for certain inhibitors on PARG glycohydrolase activity. These assays offer a complementary approach for studying target engagement within the PARylation cycle. Citation Format: Ani Michaud, Kelly Teske, Jennifer Wilkinson, Chad Zimprich, James Vasta, Cesear Corona, Min Zhou, Elizabeth Dominguez, Kaitlin Dunn-Hoffman, Matthew Robers. Exploring the landscape of PARP and PARG inhibitor selectivity in live cells using NanoBRET™ target engagement assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7114.

Poly-(ADP-ribose)polymerases inhibitionby olaparib attenuates activities of the NLRP3 inflammasome and of NF-κB in THP-1 monocytes.

Poly-(ADP-ribose) polymerases (PARPs) are a protein family that make ADP-ribose modifications on target genes and proteins. PARP family members contribute to the pathogenesis of chronic inflammatory diseases, including atherosclerosis, in which monocytes/macrophages play important roles. PARP inhibition is protective against atherosclerosis. However, the mechanisms by which PARP inhibition exerts this beneficial effect are not well understood. Here we show that in THP-1 monocytes, inhibition of PARP by olaparib attenuated oxidized low-density lipoprotein (oxLDL)-induced protein expressions of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing-3 (NLRP3) inflammasome components: NLRP3, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. Consistent with this effect, olaparib decreased oxLDL-enhanced interleukin (IL)-1β and IL-18 protein expression. Olaparib also decreased the oxLDL-mediated increase in mitochondrial reactive oxygen species. Similar to the effects of the NLRP3 inhibitor, MCC950, olaparib attenuated oxLDL-induced adhesion of monocytes to cultured human umbilical vein endothelial cells and reduced foam cell formation. Furthermore, olaparib attenuated the oxLDL-mediated activation of nuclear factor (NF)-κB through the oxLDL-mediated increase in IκBα phosphorylation and assembly of NF-κB subunits, demonstrated by co-immunoprecipitation of IκBα with RelA/p50 and RelB/p52 subunits. Moreover, PARP inhibition decreased oxLDL-mediated protein expression of a NF-κB target gene, VCAM1, encoding vascular cell adhesion molecule-1. This finding indicates an important role for NF-κB activity in PARP-mediated activation of the NLRP3 inflammasome. Thus, PARP inhibition by olaparib attenuates NF-κB and NLRP3 inflammasome activities, lessening monocyte cell adhesion and macrophage foam cell formation. These inhibitory effects of olaparib on NLRP3 activity potentially protect against atherosclerosis.

Phthalimide-tethered isatins as novel poly(ADP-ribose) polymerase inhibitors: Design, synthesis, biological evaluations, and molecular modeling investigations.

Humanity is currently facing various diseases with significant mortality rates, particularly those associated with malignancies. Numerous enzymes and proteins have been identified as highly promising targets for the treatment of cancer. The poly(ADP-ribose) polymerases (PARPs) family comprises 17 members which are essential in DNA damage repair, allowing the survival of cancer cells. Unlike other PARP family members, PARP-1 and, to a lesser extent, PARP-2 show more than 90% activity in response to DNA damage. PARP-1 levels were shown to be elevated in various tumor cells, including breast, lung, ovarian, and prostate cancer and melanomas. Accordingly, novel series of phthalimide-tethered isatins (6a-n, 10a-e, and 11a-e) were synthesized as potential PARP-1 inhibitors endowed with anticancer activity. All the synthesized molecules were assessed against PARP-1, where compounds 6f and 10d showed nanomolar activities with IC50 = 15.56 ± 2.85 and 13.65 ± 1.42 nM, respectively. Also, the assessment of the antiproliferative effects of the synthesized isatins was conducted on four cancer cell lines: leukemia (K-562), liver (HepG2), and breast (MCF-7 and HCC1937) cancers. Superiorly, compounds 6f and 10d demonstrated submicromolar IC50 values against breast cancer MCF-7 (IC50 = 0.92 ± 0.18 and 0.67 ± 0.12 µM, respectively) and HCC1937 (IC50 = 0.88 ± 0.52 and 0.53 ± 0.11 µM, respectively) cell lines. In addition, compounds 6f and 10d induced arrest in the G2/M phase of the cell cycle as compared to untreated cells. Finally, in silico studies, including docking and molecular dynamic simulations, were performed to justify the biological results.

Synergistic Leukaemia Cell Death Induced with PARP and TiP60 Inhibitors Is a Prospective Therapeutic Strategy for AML

Background: We previously identified pharmacological inhibition of lysine demethylase 4A (KDM4A) sensitises AML cells to inhibition of poly ADP ribose polymerase (PARP), one of the major DNA damage response (DDR) mediators. We hypothesise that synergy between inhibition of PARP and histone acetyltransferase (HAT) family member, Tip60 that is involved in DSB repair, may drive effective AML leukemic cell death sparing normal cells. We aim to unravel molecular mechanisms of action of the targeted inhibitors and downstream targets of Tip60 and PARP in AML. Methods: Phenotypic changes, such as in cell viability, apoptosis, cell cycle arrest by flow cytometry, colony forming capacity, and DNA damage protein markers by western blotting, in AML cells following drug treatments were determined by standard protocols. Enrichment of biological pathways in which genes with transcriptional changes in AML cells upon the drug treatment were discovered using gene set enrichment analysis (GSEA) of RNA-seq dataset generated from treated cell lines. Downstream target genes of Tip60 and PARP together in common were identified via cross-validation using least absolute shrinkage and selection operator (LASSO) linear regression in clinical datasets. Results: Loss of PARP and Tip60 enzymatic activities was driven by pharmacological inhibition of PARP and Tip60 individually and in combination in AML cell lines. As a result, a significant synergistic effect of the combination treatment (PARPi plus Tip60i) was confirmed in THP-1 and MOLM-13 cells with combination indices (CI) < 1, which was further validated via the Bliss independent model and isobolograms. Simultaneous combination of PARPi together with Tip60i resulted in significant phenotypic changes with increased early and late apoptotic cell populations, cell cycle arrest at S phase, decreased colony forming ability, and accumulation of DNA lesion markers, γH2A.X and cleaved PARP proteins, which contributed to inhibition of cell growth and ultimately significant increase (p<0.0001) in AML leukemic cell death. GSEA revealed that combination treatment of PARPi and Tip60i induced significant enrichment of genes (adjusted p-value<0.05) involved in DDR sub-pathways, mismatch repair (MMR; NES=-1.54; FDR=0.088) and interstrand crosslink repair (ICLR; NES=-1.25; FDR=0.22), in parallel, suggesting an inhibitory role of PARPi and Tip60i in impairing DDR in AML cells. Furthermore, global GSEA further demonstrated that synergistic effect of PARPi plus Tip60i induced significant upregulation of biological pathways of hypoxia, unfolded protein response, p53 pathway, and apoptosis; and significant downregulation of oxidative phosphorylation, glycolysis, and Myc pathway. A minimum subset of 13 genes mediated by inhibition of Tip60 and PARP ('TiP13') in AML cells was constructed in relation to patient survival. Kaplan-Meier survival analysis showed that the gene signature score of each patient in four patient cohorts, calculated as the sum of the weighted gene expression for each gene in TiP13, was highly correlated with patient survival in four test clinical datasets (GSE12417, FDR=0.00091; GSE37642, FDR=0.042; GSE6891, FDR=0.044; Beat AML, FDR<0.0001). Discussion: Failure to repair DNA damage at transcriptional level by blocking of MMR and ICLR pathways led to constant accumulation of DNA lesions and thereby effective cell killing in AML cells treated with PARPi and Tip60i in combination. This study further demonstrates that transcriptional outcomes driven by synergising PARPi and Tip60i activities contribute to dysregulation of oxidative phosphorylation for oxidation of nutrients and production of ATP in glycolysis in AML cells, leading to genomic instability, accumulated global DNA damage, and increased response to stress or unfolded protein. Consequently, the accumulated DNA lesions alongside impaired capacity of AML cells to repair their damaged DNA, thereby stimulated apoptosis, facilitated with downregulation of an oncogenic Myc pathway and upregulation of tumour suppressor p53 pathway. In addition, this study has uncovered a novel TiP13 gene signature (downstream targets of PARP and Tip60) with prognostic significance in AML that successfully stratifies outcomes of AML patients.

Abstract 2463: 18F-Talazoparib: A novel potential PET imaging agent of PARP

Abstract Background: Poly-(ADP-ribose) polymerases (PARPs) are an enzyme family that catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to an acceptor protein. PARP family members play fundamental roles in single-strand DNA break (SSB) repair, cell signaling of DNA damage and inflammation, cell death, and cellular replication. Various PARP inhibitors have been developed and PARP1 inhibitors are currently used to treat subsets of breast and ovarian cancer patients. Of all the PARP inhibitors studied, Talazoparib (Pfizer), appears to be the most stable in pre-clinical and clinical studies, affords nanomolar affinity to PARP1, and demonstrates the highest affinity trapping of PARP on the DNA-protein complex. Imaging of PARP using a radiolabeled inhibitor has been proposed for patient selection, therapy dose optimization and validation of target engagement. Several PARP imaging agents have been developed and studied as possible tracers, by modifying the structure of an original PARP inhibitor to accommodate an imaging or therapeutic isotope. Herein we used a copper-mediated 18F-radio-fluorination strategy with a novel aryl boronic ester precursor to access 18F-Talazoparib (18F-TZ), a 18F-radiolabeled form of the structurally identical Talazoparib. Methods: A novel boronic ester precursor was synthetized in six steps synthesis with an overall yield of 3.3% and characterized by NMR and mass spectrometry. Radiofluorination was performed using an automatic GE TracerLab system and analytical radioHPLC was used for tracer characterization and for stability analysis. A panel of tumor cell lines representing a variety of PARP expression levels (Hela, SUM149, SUM1315, MDA-MB-436, MDA-MB-231, PSN1, MiaPaca2, Jurkat) were tested for time-dependent uptake and specific binding, utilizing both 4 °C and excess cold TZ blocking. Results: 18F-TZ was synthetized in 5.0±0.80% (n=20) radiochemical yield and a molar activity of 18.9-98.0 GBq/µmol by a copper-mediated radiofluorination of the corresponding pinacolic boronic ester, followed by purification via semi-preparative HPLC and reformulation in ethanol. 18F-TZ was obtained in &amp;gt;99% radiochemical purity, displaying up to 4 hr shelf stability (25 °C and 4 °C) in PBS (10% EtOH). 18F-TZ was also stable in human plasma up to 4 hr at 37 °C. Both shelf and plasma stability tests showed a decrease of the radioHPLC peak consistent with decay half-life, but did not show the formation of secondary species. All cell lines selected for the in cellulo study showed uptake of radioactive 18F-TZ after 30 min of incubation at 37 °C. Cell uptake was blocked &amp;gt;98% by excess cold TZ or incubation of cells at 4 °C. Conclusion: 18F-Talazoparib was synthesized by copper-mediated radiofluorination from the corresponding pinacol ester and demonstrates both uptake and target specificity in PARP-expressing tumor cells. Citation Format: Riccardo Muzzioli, Federica Pisaneschi, Yi Rao, Seth Gammon, David Piwnica-Worms. 18F-Talazoparib: A novel potential PET imaging agent of PARP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2463.

Research Advances in the Role of the Poly ADP Ribose Polymerase Family in Cancer.

Poly ADP ribose polymerases (PARPs) catalyze the modification of acceptor proteins, DNA, or RNA with ADP-ribose, which plays an important role in maintaining genomic stability and regulating signaling pathways. The rapid development of PARP1/2 inhibitors for the treatment of ovarian and breast cancers has advanced research on other PARP family members for the treatment of cancer. This paper reviews the role of PARP family members (except PARP1/2 and tankyrases) in cancer and the underlying regulatory mechanisms, which will establish a molecular basis for the clinical application of PARPs in the future.

Discovery of 5-{4-[(7-Ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl]piperazin-1-yl}-N-methylpyridine-2-carboxamide (AZD5305): A PARP1-DNA Trapper with High Selectivity for PARP1 over PARP2 and Other PARPs.

Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1-DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure- and property-based design of 25 (AZD5305), a potent and selective PARP1 inhibitor and PARP1-DNA trapper with excellent in vivo efficacy in a BRCA mutant HBCx-17 PDX model. Compound 25 is highly selective for PARP1 over other PARP family members, with good secondary pharmacology and physicochemical properties and excellent pharmacokinetics in preclinical species, with reduced effects on human bone marrow progenitor cells in vitro.

PARP10 Multi-Site Auto- and Histone MARylation Visualized by Acid-Urea Gel Electrophoresis.

Poly-ADP-ribose polymerase (PARP)-family ADP-ribosyltransferases function in various signaling pathways, predominantly in the nucleus and cytosol. Although PARP inhibitors are in clinical practice for cancer therapy, the enzymatic activities of individual PARP family members are yet insufficiently understood. We studied PARP10, a mono-ADP-ribosyltransferase and potential drug target. Using acid-urea gel electrophoresis, we found that the isolated catalytic domain of PARP10 auto-ADP-ribosylates (MARylates) at eight or more acceptor residues. We isolated individual species with either singular or several modifications and then analyzed them by mass spectrometry. The results confirmed multi-site MARylation in a random order and identified four acceptor residues. The mutagenesis of singular acceptor residues had a minor impact on the overall auto-MARylation level and no effect on the MARylation of histone H3.1. Together, our results suggest that PARP10 automodification may have functions in the regulation of intramolecular or partner binding events, rather than of its enzymatic catalysis. This contributes to a better understanding of PARP10 functions, and, in the long run, to gauging the consequences of PARP inhibitor actions.

Progress and outlook in studying the substrate specificities of PARPs and related enzymes.

Despite decades of research on ADP-ribosyltransferases (ARTs) from the poly(ADP-ribose) polymerase (PARP) family, one key aspect of these enzymes - their substrate specificity - has remained unclear. Here, we briefly discuss the history of this area and, more extensively, the recent breakthroughs, including the identification of protein serine residues as a major substrate of PARP1 and PARP2 in human cells and of cysteine and tyrosine as potential targets of specific PARPs. On the molecular level, the modification of serine residues requires a composite active site formed by PARP1 or PARP2 together with a specificity-determining factor, HPF1; this represents a new paradigm not only for PARPs but generally for post-translational modification (PTM) catalysis. Additionally, we discuss the identification of DNA as a substrate of PARP1, PARP2 and PARP3, and some bacterial ARTs and the discovery of noncanonical RNA capping by several PARP family members. Together, these recent findings shed new light on PARP-mediated catalysis and caution to 'expect the unexpected' when it comes to further potential substrates.

Poly(ADP-ribose) polymerase enzymes and the maintenance of genome integrity.

DNA damage response (DDR) relies on swift and accurate signaling to rapidly identify DNA lesions and initiate repair. A critical DDR signaling and regulatory molecule is the posttranslational modification poly(ADP-ribose) (PAR). PAR is synthesized by a family of structurally and functionally diverse proteins called poly(ADP-ribose) polymerases (PARPs). Although PARPs share a conserved catalytic domain, unique regulatory domains of individual family members endow PARPs with unique properties and cellular functions. Family members PARP-1, PARP-2, and PARP-3 (DDR-PARPs) are catalytically activated in the presence of damaged DNA and act as damage sensors. Family members tankyrase-1 and closely related tankyrase-2 possess SAM and ankyrin repeat domains that regulate their diverse cellular functions. Recent studies have shown that the tankyrases share some overlapping functions with the DDR-PARPs, and even perform novel functions that help preserve genomic integrity. In this review, we briefly touch on DDR-PARP functions, and focus on the emerging roles of tankyrases in genome maintenance. Preservation of genomic integrity thus appears to be a common function of several PARP family members, depicting PAR as a multifaceted guardian of the genome.

New perspectives on the plant PARP family: Arabidopsis PARP3 is inactive, and PARP1 exhibits predominant poly (ADP-ribose) polymerase activity in response to DNA damage

BackgroundPoly (ADP-ribosyl) ation (PARylation) is an important posttranslational modification that regulates DNA repair, gene transcription, stress responses and developmental processes in multicellular organisms. Poly (ADP-ribose) polymerase (PARP) catalyzes PARylation by consecutively adding ADP-ribose moieties from NAD+ to the amino acid receptor residues on target proteins. Arabidopsis has three canonical PARP members, and two of these members, AtPARP1 and AtPARP2, have been demonstrated to be bona fide poly (ADP-ribose) polymerases and to regulate DNA repair and stress response processes. However, it remains unknown whether AtPARP3, a member that is highly expressed in seeds, has similar biochemical activity to that of AtPARP1 and AtPARP2. Additionally, although both the phylogenetic relationships and structural similarities indicate that AtPARP1 and AtPARP2 correspond to animal PARP1 and PARP2, respectively, two previous studies have indicated that AtPARP2, and not AtPARP1, accounts for most of the PARP activity in Arabidopsis, which is contrary to the knowledge that PARP1 is the predominant PARP in animals.ResultsIn this study, we obtained both in vitro and in vivo evidence demonstrating that AtPARP3 does not act as a typical PARP in Arabidopsis. Domain swapping and point mutation assays indicated that AtPARP3 has lost NAD+-binding capability and is inactive. In addition, our results showed that AtPARP1 was responsible for most of the PARP enzymatic activity in response to the DNA damage-inducing agents zeocin and methyl methanesulfonate (MMS) and was more rapidly activated than AtPARP2, which supports that AtPARP1 remains the predominant PARP member in Arabidopsis. AtPARP1 might first become activated by binding to damaged sites, and AtPARP2 is then poly (ADP-ribosyl) ated by AtPARP1 in vivo.ConclusionsCollectively, our biochemical and genetic analysis results strongly support the notion that AtPARP3 has lost poly (ADP-ribose) polymerase activity in plants and performs different functions from those of AtPARP1 and AtPARP2. AtPARP1, instead of AtPARP2, plays the predominant role in PAR synthesis in both seeds and seedlings. These data bring new insights into our understanding of the physiological functions of plant PARP family members.

Enabling drug discovery for the PARP protein family through the detection of mono-ADP-ribosylation

Poly-ADP-ribose polymerases (PARPs) are a family of enzymes responsible for transferring individual or chains of ADP-ribose subunits to substrate targets as a type of post-translational modification. PARPs regulate a wide variety of important cellular processes, ranging from DNA damage repair to antiviral response. However, most research to date has focused primarily on the polyPARPs, which catalyze the formation of ADP-ribose polymer chains, while the monoPARPs, which transfer individual ADP-ribose monomers, have not been studied as thoroughly. This is partially due to the lack of robust assays to measure mono-ADP-ribosylation in the cell. In this study, the recently developed MAR/PAR antibody has been shown to detect mono-ADP-ribosylation in cells, enabling the field to investigate the function and therapeutic potential of monoPARPs. In this study, the antibody was used in conjunction with engineered cell lines that overexpress various PARPs to establish a panel of assays to evaluate the potencies of literature-reported PARP inhibitors. These assays should be generally applicable to other PARP family members for future compound screening efforts. A convenient and generalizable workflow to identify and validate PARP substrates has been established. As an initial demonstration, aryl hydrocarbon receptor was verified as a direct PARP7 substrate and other novel substrates for this enzyme were also identified and validated. This workflow takes advantage of commercially available detection reagents and conventional mass spectrometry instrumentation and methods. Ultimately, these assays and methods will help drive research in the PARP field and benefit future therapeutics development.

PARPs in genome stability and signal transduction: implications for cancer therapy.

The poly(ADP-ribose) polymerase (PARP) superfamily of enzymes catalyses the ADP-ribosylation (ADPr) of target proteins by using nicotinamide adenine dinucleotide (NAD+) as a donor. ADPr reactions occur either in the form of attachment of a single ADP-ribose nucleotide unit on target proteins or in the form of ADP-ribose chains, with the latter called poly(ADP-ribosyl)ation. PARPs regulate many cellular processes, including the maintenance of genome stability and signal transduction. In this review, we focus on the PARP family members that possess the ability to modify proteins by poly(ADP-ribosyl)ation, namely PARP1, PARP2, Tankyrase-1, and Tankyrase-2. Here, we detail the cellular functions of PARP1 and PARP2 in the regulation of DNA damage response and describe the function of Tankyrases in Wnt-mediated signal transduction. Furthermore, we discuss how the understanding of these pathways has provided some major breakthroughs in the treatment of human cancer.

Abstract LB-273: A head-to-head comparison of the properties of five clinical PARP inhibitors identifies new insights that can explain both the observed clinical efficacy and safety profiles

Abstract Four poly(ADP-ribose) polymerase (PARP) inhibitors have now presented phase 3 monotherapy data showing compelling benefit of targeting tumours enriched with DNA damage response (DDR) pathway deficiencies, including BRCA gene mutations. Indirect treatment comparisons using the published clinical data from these late stage trials suggest similar levels of monotherapy efficacy are observed in spite of reported differences in PARP trapping potency. However, there is greater diversity in the observed safety profiles. To try and understand these observations, we have carried out a head-to-head comparison of these four PARP inhibitors (olaparib, niraparib, rucaparib and talazoparib) as well as veliparib, which recently reported phase 3 chemotherapy combination data. In our studies, we included an assessment of molecular mechanism of action that included PAR inhibition, PARP trapping and synthetic lethality in isogenic BRCA mutant and wild type models. In addition, an assessment of selectivity in terms of both inhibition of PARP family members using a novel chemoproteomic approach, as well as secondary (off-target) activities was performed. Finally, effects on human haematopoietic stem cell viability and bio-distribution to bone marrow in the rat were tested and compared. A detailed correlation of our datasets with the observed clinical results, including adverse events, suggests these preclinical experiments provide an excellent predictor of clinical response and could be used to assess emerging as well as novel PARP inhibitors. OlaparibVeliparibRucaparibNiraparibTalazoparibCompanyAZAbbVieClovisTesaroPfizerPhaseApprovedIIIApprovedApprovedIIIPARP1 SPR Kd (µM)0.0010.0070.0010.0130.002PARP2 SPR Kd (µM)0.0010.0140.0230.0430.005PARPs with Proteomic Kd &amp;lt;1 µM1,2,3,4,131,2,3,4,131,2,3,4,10,131,2,131,2,3,4,5a,13, 16Sec. Pharm. #, top hit µM0/855/85 5HT7, 0.513/85 5HT4, 0.517/84 DAT, 0.040/85Monotherapy dose (mg)300 bd (tablet)500 bd600 bd300 od1 od Citation Format: Elisabetta Leo, Jeffrey Johannes, Giuditta Illuzzi, Andrew Zhang, Paul Hemsley, Michal J. Bista, Jonathan P. Orme, Verity A. Talbot, Ana J. Narvaez, Elizabeth Underwood, Andrew Pike, Jenni K. Nikkila, Lucy Riches, Sinbad Sweeney, Frida Gustafsson, Anna Cronin, Piero Ricchiuto, Debora A. Roaquin, Fiona Pachl, Eric Miele, Ruth MacDonald, Glen Hawthorne, Andrew N. Mead, Mark J. O'Connor. A head-to-head comparison of the properties of five clinical PARP inhibitors identifies new insights that can explain both the observed clinical efficacy and safety profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-273.

PARP10 (ARTD10) modulates mitochondrial function.

Poly(ADP-ribose) polymerase (PARP)10 is a PARP family member that performs mono-ADP-ribosylation of target proteins. Recent studies have linked PARP10 to metabolic processes and metabolic regulators that prompted us to assess whether PARP10 influences mitochondrial oxidative metabolism. The depletion of PARP10 by specific shRNAs increased mitochondrial oxidative capacity in cellular models of breast, cervical, colorectal and exocrine pancreas cancer. Upon silencing of PARP10, mitochondrial superoxide production decreased in line with increased expression of antioxidant genes pointing out lower oxidative stress upon PARP10 silencing. Improved mitochondrial oxidative capacity coincided with increased AMPK activation. The silencing of PARP10 in MCF7 and CaCo2 cells decreased the proliferation rate that correlated with increased expression of anti-Warburg enzymes (Foxo1, PGC-1α, IDH2 and fumarase). By analyzing an online database we showed that lower PARP10 expression increases survival in gastric cancer. Furthermore, PARP10 expression decreased upon fasting, a condition that is characterized by increases in mitochondrial biogenesis. Finally, lower PARP10 expression is associated with increased fatty acid oxidation.

Identifying Genomic Sites of ADP-Ribosylation Mediated by Specific Nuclear PARP Enzymes Using Click-ChIP.

Nuclear poly(ADP-ribose) polymerases (PARPs), including PARPs 1, 2, and 3 and the Tankyrases, belong to a family of enzymes that can bind to chromatin and covalently modify histone- and chromatin-associated proteins with ADP-ribose derived from nuclear NAD+. The genomic loci where the nuclear PARPs bind and covalently modify chromatin are a fundamental question in PARP biology. Chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) has become an essential tool for determining specific sites of binding and modification genome-wide. Few methods are available, however, for localizing PARP-specific ADP-ribosylation events across the genome. Here we describe a variation of ChIP-seq, called Click-ChIP-seq, for identifying sites of ADP-ribosylation mediated by specific PARP family members. This method uses analog-sensitive PARP (asPARP) technology, including asPARP mutants and the alkyne-containing "clickable" NAD+ analog 8-Bu(3-yne)T-NAD+. In this assay, nuclei from cells expressing an asPARP protein of interest are incubated with 8-Bu(3-yne)T-NAD+, which is incorporated into ADP-ribose modifications mediated only by that specific asPARP protein. The nuclei are then subjected to cross-linking with formaldehyde, and the protein-linked analog ADP-ribose is clicked to biotin using copper-catalyzed alkyne-azide "click" chemistry. The chromatin is fragmented, and the fragments containing analog ADP-ribose are enriched using streptavidin-mediated precipitation. Finally, the enriched DNA is analyzed by qPCR or deep-sequencing experiments to determine which genomic loci contain ADP-ribose modifications mediated by the specific PARP protein of interest. Click-ChIP-seq has proven to be a robust and reproducible method for identifying chromatin-associated, PARP-specific ADP-ribosylation events genome-wide.

RNAi-mediated knockdown of Parp1 does not improve the development of female cloned mouse embryos.

Somatic cell nuclear transfer is an important technique for life science research, but its efficiency is still extremely low, and most genes that are important during early development, such as X chromosome-linked genes, are not appropriately expressed during this process. Poly (ADP-ribose) polymerase (PARP) is an enzyme that transfers ADP ribose clusters to target proteins. PARP family members such as PARP1 participate in cellular signalling pathways through poly (ADP-ribosylation) (PARylation), which ultimately promotes changes in chromatin structure, gene expression, and the localization and activity of proteins that mediate signalling responses. PARP1 is associated with X chromosome inactivation (Xi). Here, we showed that abnormal Xi occurs in somatic cell nuclear transfer (NT) blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. Parp1 expression was higher in female NT blastocysts than that in intracytoplasmic sperm injection (ICSI) embryos but not in male NT blastocysts. After knocking down Parp1 expression, both the pre-rRNA 47S and X-inactivation-specific transcript (Xist) levels increased. Moreover, the expression of genes on the inactivated X chromosome, such as Magea6 and Msn, were also increased in the NT embryos. However, the development of Parp1si NT embryos was impaired, although total RNA sequencing showed that overall gene expression between the Parp1si NT blastocysts and the control was similar. Our findings demonstrate that increases in the expression of several genes on the X chromosome and of rRNA primary products in NT blastocysts with disrupted Parp1 expression are insufficient to rescue the impaired development of female cloned mouse embryos and could even exacerbate the associated developmental deficiencies.

PARP activity and inhibition in fetal and adult oligodendrocyte precursor cells: Effect on cell survival and differentiation

Poly (ADP-ribose) polymerase (PARP) family members are ubiquitously expressed and play a key role in cellular processes, including DNA repair and cell death/survival balance. Accordingly, PARP inhibition is an emerging pharmacological strategy for cancer and neurodegenerative diseases. Consistent evidences support the critical involvement of PARP family members in cell differentiation and phenotype maturation. In this study we used an oligodendrocyte precursor cells (OPCs) enriched system derived from fetal and adult brain to investigate the role of PARP in OPCs proliferation, survival, and differentiation. The PARP inhibitors PJ34, TIQ-A and Olaparib were used as pharmacological tools. The main results of the study are: (i) PARP mRNA expression and PARP activity are much higher in fetal than in adult-derived OPCs; (ii) the culture treatment with PARP inhibitors is cytotoxic for OPCs derived from fetal, but not from adult, brain; (iii) PARP inhibition reduces cell number, according to the inhibitory potency of the compounds; (iv) PARP inhibition effect on fetal OPCs is a slow process; (v) PARP inhibition impairs OPCs maturation into myelinating OL in fetal, but not in adult cultures, according to the inhibitory potency of the compounds. These results have implications for PARP-inhibition therapies for diseases and lesions of the central nervous system, in particular for neonatal hypoxic/ischemic encephalopathy.