PARP and PARG inhibitors in cancer treatment.

Oxidative and replication stress underlie genomic instability of cancer cells. Amplifying genomic instability through radiotherapy and chemotherapy has been a powerful but nonselective means of killing cancer cells. Precision medicine has revolutionized cancer therapy by putting forth the concept of selective targeting of cancer cells. Poly(ADP-ribose) polymerase (PARP) inhibitors represent a successful example of precision medicine as the first drugs targeting DNA damage response to have entered the clinic. PARP inhibitors act through synthetic lethality with mutations in DNA repair genes and were approved for the treatment of BRCA mutated ovarian and breast cancer. PARP inhibitors destabilize replication forks through PARP DNA entrapment and induce cell death through replication stress-induced mitotic catastrophe. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) exploit and exacerbate replication deficiencies of cancer cells and may complement PARP inhibitors in targeting a broad range of cancer types with different sources of genomic instability. Here I provide an overview of the molecular mechanisms and cellular consequences of PARP and PARG inhibition. I highlight clinical performance of four PARP inhibitors used in cancer therapy (olaparib, rucaparib, niraparib, and talazoparib) and discuss the predictive biomarkers of inhibitor sensitivity, mechanisms of resistance as well as the means of overcoming them through combination therapy.

Chemical genetic methodologies for identifying protein substrates of PARPs.

Poly-ADP-ribose-polymerases (PARPs) are a family of 17 enzymes that regulate a diverse range of cellular processes in mammalian cells. PARPs catalyze the transfer of ADP-ribose from NAD+ to target molecules, most prominently amino acids on protein substrates, in a process known as ADP-ribosylation. Identifying the direct protein substrates of individual PARP family members is an essential first step for elucidating the mechanism by which PARPs regulate a particular pathway in cells. Two distinct chemical genetic (CG) strategies have been developed for identifying the direct protein substrates of individual PARP family members. In this review, we discuss the design principles behind these two strategies and how target identification has provided novel insight into the cellular function of individual PARPs and PARP-mediated ADP-ribosylation.

Apurinic/apyrimidinic endonuclease 1 has major impact in prevention of suicidal covalent DNA-protein crosslink with apurinic/apyrimidinic site in cellular extracts.

DNA-protein crosslinks (DPC) are common DNA lesions induced by various external and endogenous agents. One of the sources of DPC is the apurinic/apyrimidinic site (AP site) and proteins interacting with it. Some proteins possessing AP lyase activity form covalent complexes with AP site-containing DNA without borohydride reduction (suicidal crosslinks). We have shown earlier that tyrosyl-DNA phosphodiesterase 1 (TDP1) but not AP endonuclease 1 (APE1) is able to remove intact OGG1 from protein-DNA adducts, whereas APE1 is able to prevent the formation of DPC by hydrolyzing the AP site. Here we demonstrate that TDP1 can remove intact PARP2 but not XRCC1 from covalent enzyme-DNA adducts with AP-DNA formed in the absence of APE1. We also analyzed an impact of APE1 and TDP1 on the efficiency of DPC formation in APE1-/- or TDP1-/- cell extracts. Our data revealed that APE1 depletion leads to increased levels of PARP1-DNA crosslinks, whereas TDP1 deficiency has little effect on DPC formation.

Poly ADP-ribose polymerase-1 promotes seed-setting rate by facilitating gametophyte development and meiosis in rice (Oryza sativa L.).

Poly(ADP-ribose) polymerases (PARPs), which transfer either monomer or polymer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+ ) onto target proteins, are required for multiple processes in DNA damage repair, cell cycle, development, and abiotic stress in animals and plants. Here, the uncharacterized rice (Oryza sativa) OsPARP1, which has been predicted to have two alternative OsPARP1 mRNA splicing variants, OsPARP1.1 and OsPARP1.2, was investigated. However, bimolecular fluorescence complementation showed that only OsPARP1.1 interacted with OsPARP3 paralog, suggesting that OsPARP1.1 is a functional protein in rice. OsPARP1 was preferentially expressed in the stamen primordial and pollen grain of mature stamen during flower development. The osparp1 mutant and CRISPR plants were delayed in germination, indicating that defective DNA repair machinery impairs early seed germination. The mutant displayed a normal phenotype during vegetative growth but had a lower seed-setting rate than wild-type plants under normal conditions. Chromosome bridges and DNA fragmentations were detected in male meiocytes at anaphase I to prophase II. After meiosis II, malformed tetrads or tetrads with micronuclei were formed. Meanwhile, the abnormality was also found in embryo sac development. Collectively, these results suggest that OsPARP1 plays an important role in mediating response to DNA damage and gametophyte development, crucial for rice yield in the natural environment.

NAD+, Sirtuins and PARPs: enhancing oocyte developmental competence.

Oocyte quality is the limiting factor in female fertility. It is well known that maternal nutrition plays a role in reproductive function, and manipulating nutrition to improve fertility in livestock has been common practice in the past, particularly with respect to negative energy balance in cattle. A deficiency in nicotinamide adenine dinucleotide (NAD+) production has been associated with increased incidences of miscarriage and congenital defects in humans and mice, while elevating NAD+ through dietary supplements in aged subjects improved oocyte quality and embryo development. NAD+ is consumed by Sirtuins and poly-ADP-ribose polymerases (PARPs) within the cell and thus need constant replenishment in order to maintain various cellular functions. Sirtuins and PARPs play important roles in oocyte maturation and embryo development, and their activation may prove beneficial to in vitro embryo production and livestock breeding programs. This review examines the roles of NAD+, Sirtuins and PARPs in aspects of fertility, providing insights into the potential use of NAD+-elevating treatments in livestock breeding and embryo production programs.

A Double-Edged Sword: The Two Faces of PARylation.

Poly ADP-ribosylation (PARylation) is a post-translational modification process. Following the discovery of PARP-1, numerous studies have demonstrated the role of PARylation in the DNA damage and repair responses for cellular stress and DNA damage. Originally, studies on PARylation were confined to PARP-1 activation in the DNA repair pathway. However, the interplay between PARylation and DNA repair suggests that PARylation is important for the efficiency and accuracy of DNA repair. PARylation has contradicting roles; however, recent evidence implicates its importance in inflammation, metabolism, and cell death. These differences might be dependent on specific cellular conditions or experimental models used, and suggest that PARylation may play two opposing roles in cellular homeostasis. Understanding the role of PARylation in cellular function is not only important for identifying novel therapeutic approaches; it is also essential for gaining insight into the mechanisms of unexplored diseases. In this review, we discuss recent reports on the role of PARylation in mediating diverse cellular functions and homeostasis, such as DNA repair, inflammation, metabolism, and cell death.

Arabidopsis sirtuins and poly(ADP-ribose) polymerases regulate gene expression in the day but do not affect circadian rhythms.

Nicotinamide-adenine dinucleotide (NAD) is involved in redox homeostasis and acts as a substrate for NADases, including poly(ADP-ribose) polymerases (PARPs) that add poly(ADP-ribose) polymers to proteins and DNA, and sirtuins that deacetylate proteins. Nicotinamide, a by-product of NADases increases circadian period in both plants and animals. In mammals, the effect of nicotinamide on circadian period might be mediated by the PARPs and sirtuins because they directly bind to core circadian oscillator genes. We have investigated whether PARPs and sirtuins contribute to the regulation of the circadian oscillator in Arabidopsis. We found no evidence that PARPs and sirtuins regulate the circadian oscillator of Arabidopsis or are involved in the response to nicotinamide. RNA-seq analysis indicated that PARPs regulate the expression of only a few genes, including FLOWERING LOCUS C. However, we found profound effects of reduced sirtuin 1 expression on gene expression during the day but not at night, and an embryo lethal phenotype in knockouts. Our results demonstrate that PARPs and sirtuins are not associated with NAD regulation of the circadian oscillator and that sirtuin 1 is associated with daytime regulation of gene expression.

Molecular Tools for the Study of ADP-Ribosylation: A Unified and Versatile Method to Synthesise Native Mono-ADP-Ribosylated Peptides.

ADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr in DNA damage response, whereas the physiological impact of less common ADPr-modifications of cysteine and threonine side chains is less clear. Generally, gaining molecular insights into ADPr recognition and turn-over is hampered by the availability of homogeneous, ADP-ribosylated material, such as mono-ADP-ribosylated (MARylated) peptides. Here, a new and efficient solid-phase strategy for the synthesis of Ser-, Thr- and Cys-MARylated peptides is described. ADP-ribosylated cysteine, apart from being a native post-translational modification in its own right, proved to be suitable as a stabile bioisostere for ADP-ribosylated serine making it a useful tool to further biochemical research on serine ADP-ribosylation. In addition, it was discovered that the Streptococcus pyogenes encoded protein, SpyMacroD, acts as a Cys-(ADP-ribosyl) hydrolase.

Active Targeted Nanoparticles for Delivery of Poly(ADP-ribose) Polymerase (PARP) Inhibitors: A Preliminary Review.

Nanotechnology has revolutionized novel drug delivery strategies through establishing nanoscale drug carriers, such as niosomes, liposomes, nanomicelles, dendrimers, polymeric micelles, and nanoparticles (NPs). Owing to their desirable cancer-targeting efficacy and controlled release, these nanotherapeutic modalities are broadly used in clinics to improve the efficacy of small-molecule inhibitors. Poly(ADP-ribose) polymerase (PARP) family members engage in various intracellular processes, including DNA repair, gene transcription, signal transduction, cell cycle regulation, cell division, and antioxidant response. PARP inhibitors are synthetic small-molecules that have emerged as one of the most successful innovative strategies for targeted therapy in cancer cells harboring mutations in DNA repair genes. Despite these advances, drug resistance and unwanted side effects are two significant drawbacks to using PARP inhibitors in the clinic. Recently, the development of practical nanotechnology-based drug delivery systems has tremendously improved the efficacy of PARP inhibitors. NPs can specifically accumulate in the leaky vasculature of the tumor and cancer cells and release the chemotherapeutic moiety in the tumor microenvironment. On the contrary, NPs are usually unable to permeate across the body's normal organs and tissues; hence the toxicity is zero to none. NPs can modify the release of encapsulated drugs based on the composition of the coating substance. Delivering PARP inhibitors without modulation often leads to the toxic effect; therefore, a delivery vehicle is essential to encapsulate them. Various nanocarriers have been exploited to deliver PARP inhibitors in different cancers. Through this review, we hope to cast light on the most innovative advances in applying PARP inhibitors for therapeutic purposes.

PARP Power: A Structural Perspective on PARP1, PARP2, and PARP3 in DNA Damage Repair and Nucleosome Remodelling.

Poly (ADP-ribose) polymerases (PARP) 1-3 are well-known multi-domain enzymes, catalysing the covalent modification of proteins, DNA, and themselves. They attach mono- or poly-ADP-ribose to targets using NAD+ as a substrate. Poly-ADP-ribosylation (PARylation) is central to the important functions of PARP enzymes in the DNA damage response and nucleosome remodelling. Activation of PARP happens through DNA binding via zinc fingers and/or the WGR domain. Modulation of their activity using PARP inhibitors occupying the NAD+ binding site has proven successful in cancer therapies. For decades, studies set out to elucidate their full-length molecular structure and activation mechanism. In the last five years, significant advances have progressed the structural and functional understanding of PARP1-3, such as understanding allosteric activation via inter-domain contacts, how PARP senses damaged DNA in the crowded nucleus, and the complementary role of histone PARylation factor 1 in modulating the active site of PARP. Here, we review these advances together with the versatility of PARP domains involved in DNA binding, the targets and shape of PARylation and the role of PARPs in nucleosome remodelling.

Soft tissue sarcomas: new opportunity of treatment with PARP inhibitors?

BACKGROUND: Poly(ADP-ribose) polymerases (PARP) are a large family of enzymes involved in several cellular processes, including DNA single-strand break repair via the base-excision repair pathway. PARP inhibitors exert antitumor activity by both catalytic PARP inhibition and PARP-DNA trapping, moreover PARP inhibition represents a potential synthetic lethal approach against cancers with specific DNA-repair defects. Soft tissue sarcoma (STSs) are a heterogeneous group of mesenchymal tumors with locally destructive growth, high risk of recurrence and distant metastasis. OBJECTIVES: The purpuse of this review is to provide an overview of the main preclinical and clinical data on use of PARPi in STSs and of effect and safety of combination of PARPi with irradiation. RESULTS: Due to numerous genomic alterations in STSs, the DNA damage response pathway can offer an interesting target for biologic therapy. Preclinical and clinical studies showed promising results, with the most robust evidences of PARPi efficacy obtained on Ewing sarcoma bearing EWS-FLI1 or EWS-ERG genomic fusions. The activity of PARP inhibitors resulted potentiated by chemotherapy and radiation. Although mechanisms of synergisms are not completely known, combination of radiation therapy and PARP inhibitors exerts antitumor effect by accumulation of unrepaired DNA damage, arrest in G2/M, activity both on oxic and hypoxic cells, reoxygenation by effect on vessels and promotion of senescence. Early trials have shown a good tolerance profile. CONCLUSIONS: The use of PARP inhibitors in advanced stage STSs, alone or combined in multimodal treatments, is of great interest and warrants further investigations.

Where Do We Stand on the Integration of PARP Inhibitors for the Treatment of Breast Cancer?

PURPOSE OF REVIEW: To provide an overview of the clinical development of poly(ADP-ribose) polymerase inhibitors (PARPi) in breast cancer to date and to review existing challenges and future research directions. RECENT FINDINGS: We summarize the clinical development of PARPi in breast cancer from bench to bedside, and discuss the results of recent phase 3 trials in patients with metastatic breast cancer (MBC) and germline mutations in BRCA1/2 (gBRCAm). We will also provide an update regarding mechanisms of action and resistance to PARPi, and review clinical trials of PARPi as monotherapy or in combination regimens. PARPi are a novel treatment approach in persons with gBRCA1/2m-associated MBC. Going forward, the clinical applicability of these compounds outside the gBRCAm setting will be studied in greater detail. The identification of accurate predictive biomarkers of response is a research priority.

A Review on DNA Repair Inhibition by PARP Inhibitors in Cancer Therapy.

The DNA repair process protects the cells from DNA damaging agent by multiple pathways. Majority of the cancer therapy cause DNA damage which leads to apoptosis. The cell has natural ability to repair this damage which ultimately leads to development of resistance of drugs. The key enzymes involved in DNA repair process are poly(ADP-ribose) (PAR) and poly(ADP-ribose) polymerases (PARP). Tumor cells repair their defective gene via defective homologues recombination (HR) in the presence of enzyme PARP. PARP inhibitors inhibit the enzyme poly(ADP-ribose) polymerases (PARPs) which lead to apoptosis of cancer cells. Current clinical data shows the role of PARP inhibitors is not restricted to BRCA mutations but also effective in HR dysfunctions related tumors. Therefore, investigation in this area could be very helpful for future therapy of cancer. This review gives detail information on the role of PARP in DNA damage repair, the role of PARP inhibitors and chemistry of currently available PARP inhibitors.

LRP1 Regulates Retinal Angiogenesis by Inhibiting PARP-1 Activity and Endothelial Cell Proliferation.

OBJECTIVE: We recently demonstrated that low-density lipoprotein receptor-related protein 1 (LRP1) is required for cardiovascular development in zebrafish. However, what role LRP1 plays in angiogenesis remains to be determined. To better understand the role of LRP1 in endothelial cell function, we investigated how LRP1 regulates mouse retinal angiogenesis. APPROACH AND RESULTS: Depletion of LRP1 in endothelial cells results in increased retinal neovascularization in a mouse model of oxygen-induced retinopathy. Specifically, retinas in mice lacking endothelial LRP1 have more branching points and angiogenic sprouts at the leading edge of the newly formed vasculature. Increased endothelial proliferation as detected by Ki67 staining was observed in LRP1-deleted retinal endothelium in response to hypoxia. Using an array of biochemical and cell biology approaches, we demonstrate that poly(ADP-ribose) polymerase-1 (PARP-1) directly interacts with LRP1 in human retinal microvascular endothelial cells. This interaction between LRP1 and PARP-1 decreases under hypoxic condition. Moreover, LRP1 knockdown results in increased PARP-1 activity and subsequent phosphorylation of both retinoblastoma protein and cyclin-dependent kinase 2, which function to promote cell cycle progression and angiogenesis. CONCLUSIONS: Together, these data reveal a pivotal role for LRP1 in endothelial cell proliferation and retinal neovascularization induced by hypoxia. In addition, we demonstrate for the first time the interaction between LRP1 and PARP-1 and the LRP1-dependent regulation of PARP-1-signaling pathways. These data bring forth the possibility of novel therapeutic approaches for pathological angiogenesis.

PARP inhibitors: review of mechanisms of action and BRCA1/2 mutation targeting.

Poly(ADP-ribose) polymerases have shown true promise in early clinical studies due to reported activity in BRCA-associated cancers. PARP inhibitors may represent a potentially important new class of chemotherapeutic agents directed at targeting cancers with defective DNA-damage repair. In order to widen the prospective patient population that would benefit from PARP inhibitors, predictive biomarkers based on a clear understanding of the mechanism of action are required. In addition, a more sophisticated understanding of the toxicity profile is required if PARP inhibitors are to be employed in the curative, rather than the palliative, setting. PARP inhibitors have successfully moved into clinical practice in the past few years, with approval granted from the Food and Drug Administration (FDA) and European Medicines Agency (EMA) within the past two years. The United States FDA approval of olaparib applies to fourth-line treatment in germline BRCA-mutant ovarian cancer, and European EMA approval of olaparib for maintenance therapy in both germline and somatic BRCA-mutant platinum-sensitive ovarian cancer. This review covers the current understanding of PARP, its inhibition, and the basis of the excitement surrounding these new agents. It also evaluates future approaches and directions required to achieve full understanding of the intricate interplay of these agents at the cellular level.

Poly(ADP-ribose) polymerases in double-strand break repair: focus on PARP1, PARP2 and PARP3.

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins catalysed by Poly(ADP-ribose) polymerases (PARP). A wealth of recent advances in the biochemical and functional characterization of the DNA-dependent PARP family members have highlighted their key contribution in the DNA damage response network, the best characterized being the role of PARP1 and PARP2 in the resolution of single-strand breaks as part of the BER/SSBR process. How PARylation contributes to the repair of double-strand breaks is less well defined but has become recently the subject of significant research in the field. The aim of this review is to provide an overview of the current knowledge concerning the role of the DNA-activated PARP1, PARP2 and PARP3 in cellular response to double-strand breaks (DSB). In addition, we outline the biological significance of these properties in response to programmed DNA lesions formed during physiological processes such as antibody repertoire assembly and diversification.

PARP inhibitors.

Poly (ADP-ribose) polymerases, abbreviated as PARPs, are a group of familiar proteins that play a central role in DNA repair employing the base excision repair (BER) pathway. There about 17 proteins in this family out of which the primary nuclear PARPs are PARP-1, PARP-2, PARP-3, and tankyrases 1 and 2 (PARP-5a and -5b) .The PARP family members are known to engage in a wide range of cellular activities, for example, DNA repair, transcription, cellular signaling, cell cycle regulation and mitosis amongst others. The chief functional units of PARP-1 are an amino terminal DNA binding domain (DBD), a central auto modification domain (AMD), and a carboxyl-terminal catalytic domain (CD). PARP inhibitors are currently undergoing clinical trials as targeted treatment modalities of breast, uterine, colorectal and ovarian cancer. This review summarizes current insights into the mechanism of action of PARP inhibitors, its recent clinical trials, and potential next steps in the evaluation of this promising class of anti-cancer drugs.

[Novel strategies of ovarian cancer treatment]. / Nowe strategie leczenia raka jajnika.

Ovarian cancer is the most lethal gynecological malignancy. Due to scarce specific symptoms, women usually seek medical help once the disease is highly advanced, with distant metastases. Currently no screening is available, making this particular cancer hard to detect in the early stage. Standard treatment is insufficient for many patients, especially in the recurrent disease. This fact explains the tremendous need to search for novel therapeutic approaches. Inhibition of angiogenesis and destruction of cancer stem cells are attempts that affect the tumor microenvironment. There is a lot of potential in inhibiting poly(ADP-rybose)polymerase (PARP) or I class histone deacetylase. Drug repositioning may also be beneficial, as it gives old drugs new purposes. Metformin, a well-known antidiabetic agent, is an example of this phenomenon. Constant progress in medicine and science makes us hope for positive outcomes while treating this highly dangerous disease.

Rapamycin-resistant poly (ADP-ribose) polymerase-1 overexpression is a potential therapeutic target in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a female-predominant cystic lung disease that can lead to respiratory failure. LAM cells typically have inactivating tuberous sclerosis complex 2 (TSC2) mutations and mammalian target of rapamycin (mTOR) complex (mTORC) 1 activation. Clinical response to the mTORC1 inhibitors has been limited, prompting a search for additional therapy for LAM. In this study, we investigated the impact of TSC2 on the expression of poly (ADP-ribose) polymerase (PARP)-1 that initiates the DNA repair pathway, and tested the efficacy of PARP1 inhibitors in the survival of TSC2-deficient (TSC2(-)) cells. We analyzed publicly available expression arrays of TSC2(-) cells and validated the findings using real-time RT-PCR, immunoblotting, and immunohistochemistry. We examined the impact of rapamycin and Torin 1 on PARP1 expression. We also tested the effect of PARP1 inhibitors, 8-hydroxy-2-methylquinazoline-4-one and 3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline, on the survival of TSC2(-) cells. We identified the up-regulation of PARP1 in TSC2(-) cells relative to cells in which wild-type TSC2 has been reintroduced (TSC2-addback [TSC2(+)] cells). The transcript levels of PARP1 in TSC2(-) cells were not affected by rapamycin. PARP1 levels were increased in TSC2(-) cells, xenograft tumors of rat-derived TSC2(-) cells, renal cystadenomas from Tsc2(+/-) mice, and human LAM nodules. RNA interference of mTOR failed to reduce PARP1 levels. Proliferation and survival of TSC2(-) cells was reduced in response to PARP1 inhibitor treatment, more so than TSC2(+) cells. TSC2(-) cells exhibit higher levels of PARP1 relative to TSC2(+) cells in an mTOR-insensitive manner. PARP1 inhibitors selectively suppress the growth and induce apoptosis of TSC2(-) cells from patients with LAM. Targeting PARP1 may be beneficial in the treatment of LAM and other neoplasm with mTORC1 activation.

Chelating intracellularly accumulated zinc decreased ischemic brain injury through reducing neuronal apoptotic death.

BACKGROUND AND PURPOSE: Zinc has been reported to possess both neurotoxic and neuroprotective capabilities. The effects of elevated intracellular zinc accumulation following transient focal cerebral ischemia remain to be fully elucidated. Here, we investigated whether removing zinc with the membrane-permeable zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), would decrease the intracellular levels of zinc in the ischemic tissue, leading to reduced brain damage and improved neurological outcomes. METHODS: Rats were pretreated with TPEN or vehicle before or after a 90-minute middle cerebral artery occlusion. Cerebral infarct volume, neurological functions, neuronal apoptosis, poly(ADP-ribose) polymerase activity, and cytosolic labile zinc were assessed after ischemia and reperfusion. RESULTS: Cerebral ischemia caused a dramatic cytosolic labile zinc accumulation in the ischemic tissue, which was decreased markedly by TPEN (15 mg/kg) pretreatment. Chelating zinc lead to reduced infarct volume compared with vehicle-treated middle cerebral artery occlusion rats, accompanied by much improved neurological assessment and motor function, which were sustained for 14 days after reperfusion. We also determined that reducing zinc accumulation rescued neurons from ischemia-induced apoptotic death by reducing poly(ADP-ribose) polymerase-1 activation. CONCLUSIONS: Ischemia-induced high accumulation of intracellular zinc significantly contributed to ischemic brain damage through promotion of neuronal apoptotic death. Removing zinc may be an effective and novel approach to reduce ischemic brain injury.