Poly ADP-ribose Polymerase Activity Research Articles

A decrease in NAMPT activity impairs basal PARP-1 activity in cytidine deaminase deficient-cells, independently of NAD+

Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD+ concentration. We therefore hypothesized that defects of the NAD+ salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD+ levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.

Nicotinamide, a Poly [ADP-Ribose] Polymerase 1 (PARP-1) Inhibitor, as an Adjunctive Therapy for the Treatment of Alzheimer's Disease.

Nicotinamide (vitamin B3) is a key component in the cellular production of Nicotinamide Adenine Dinucleotide (NAD) and has long been associated with neuronal development, survival and death. Numerous data suggest that nicotinamide may offer therapeutic benefits in neurodegenerative disorders, including Alzheimer’s Disease (AD). Beyond its effect in NAD+ stores, nicotinamide is an inhibitor of Poly [ADP-ribose] polymerase 1 (PARP-1), an enzyme with multiple cellular functions, including regulation of cell death, energy/metabolism and inflammatory response. PARP-1 functions as a DNA repair enzyme but under intense DNA damage depletes the cell of NAD+ and ATP and leads to a non-apoptotic type of cell death called Parthanatos, which has been associated with the pathogenesis of neurodegenerative diseases. Moreover, NAD+ availability might potentially improve mitochondrial function, which is severely impaired in AD. PARP-1 inhibition may also exert a protective effect against neurodegeneration by its action to diminish neuroinflammation and microglial activation which are also implicated in the pathogenesis of AD. Here we discuss the evidence supporting the use of nicotinamide as adjunctive therapy for the treatment of early stages of AD based on the inhibitory effect of nicotinamide on PARP-1 activity. The data support evaluating nicotinamide as an adjunctive treatment for AD at early stages of the disease not only to increase NAD+ stores but as a PARP-1 inhibitor, raising the hypothesis that other PARP-1 inhibitors – drugs that are already approved for breast cancer treatment – might be explored for the treatment of AD.

Poly(ADP-ribosyl)ation enhances HuR oligomerization and contributes to pro-inflammatory gene mRNA stabilization

Poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification mainly catalyzed by poly-ADP-ribose polymerase 1 (PARP1). In addition to having important roles in DNA damage detection and repair, it functions in gene expression regulation, especially at the posttranscriptional level. Embryonic lethal abnormal vision-like 1/human antigen R (ELAVL/HuR), a canonical 3′ untranslated region AU-rich element-binding protein, is a crucial mRNA-stabilizing protein that protects target mRNAs from RNA-destabilizing protein- or microRNA-induced silencing complex (miRISC)-mediated degradation. Additionally, in some cases, HuR itself either promotes or suppresses translation. Here, we demonstrated that in response to inflammatory stimuli, the PARylation of HuR, mostly at the conserved D226 site, by PARP1 increased the formation of the HuR oligomer/multimer, and HuR oligomerization promoted the disassociation of miRISC and stabilized the pro-inflammatory gene mRNAs. The prevention of PARP1 activation or HuR oligomerization attenuated lipopolysaccharide-induced inflammatory gene expression and the airway recruitment of neutrophils in mouse lungs. The present study verified a novel mechanism of PARP1 and HuR PARylation in the RNA stability regulation, increasing our understanding of how PARP1 regulates gene expression.

Abstract 5210: Novel SIK2 inhibitors sensitize ovarian and breast cancer to PARP inhibitors

Abstract Genomic instability is a recognized hallmark of cancer. Germline mutations in critical DNA-repair and DNA-damage response genes predispose to cancer development, but also create vulnerabilities that can be exploited for cancer therapy. Poly (ADP-ribose) polymerase (PARP) inhibitors are selectively active in cells with homologous recombination deficiency (HRD) caused by mutations in BRCA1, BRCA2, and other DNA repair genes. PARP inhibitors elicit significant responses in ovarian and breast cancers from BRCA1 or BRCA2 mutation carriers. However, many cancers that initially respond to PARP inhibitors eventually develop drug resistance. Thus, it is important to develop new strategies to enhance PARP inhibitor sensitivity and to increase the duration of response. We have identified Salt Induced Kinase 2 (SIK2) inhibitors (ARN3236 and ARN3261) that induce double strand breaks (DSBs) in DNA of HR-competent cells and produce synthetic lethality with multiple PARP inhibitors. SIK2 is an AMP-activated protein kinase (AMPK)-related protein kinase that is required for ovarian cancer cell proliferation and metastasis. SIK2 is overexpressed and correlates with poor prognosis in patients with high-grade serous ovarian carcinoma and triple negative breast cancer. SIK2 inhibition enhances paclitaxel sensitivity in both cancer types. We have demonstrated that olaparib-induced-growth inhibition was significantly enhanced by concurrent treatment with either ARN3236 or ARN3261 in each of 12 ovarian and breast cancer cell lines tested, but not in 3 non-tumorigenic cell lines. Co-administration of olaparib with SIK2 inhibitors suppressed tumor growth and increased the survival of mice with human ovarian (OC316) and breast (MDA-MB-231) cancer xenografts without affecting animal weight. ARN3261 produced little toxicity in preclinical toxicology studies. SIK2 inhibitors decrease the phosphorylation of class-IIa HDAC4/5/7 and abolish class-IIa HDAC 4/5/7-associated transcriptional activity of Myocyte Enhancer Factor 2D (MEF2D). Genome-wide chromatin immunoprecipitation (CHIP) sequencing revealed that SIK2 inhibitors reduce MEF2D binding to regulatory regions with high-chromatin accessibility in DNA repair genes, including FANCD2, EXO1 and XRCC4, resulting in repression of critical genes in DNA DSB repair pathway and induction of apoptosis. In addition, SIK2 inhibitors significantly decreased olaparib-mediated PARP activity and enhanced olaparib-induced cytotoxicity. Together, our data argue that the combination of a SIK2 inhibitor and a PARP inhibitor has the potential to increase the magnitude and duration of PARP inhibitor activity with tolerable toxicity. Use of a SIK2 inhibitor in combination with a PARP inhibitor provides a novel therapeutic strategy for ovarian and triple negative breast cancers with or without BRCA gene mutation. Citation Format: Zhen Lu, Weiqun Mso, Janice M. Santiago-O'Farrill, Hailing Yang, Lan Pang, Ahmed A. Ahmed, Hariprasad Vankayalapati, Robert C. Bast Jr. Novel SIK2 inhibitors sensitize ovarian and breast cancer to PARP inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5210.

Poly(ADP-Ribose) Polymerase Activity and Coronary Artery Disease in Type 2 Diabetes Mellitus: An Observational and Bidirectional Mendelian Randomization Study.

Experimental evidence suggests a close link between PARP (poly[ADP-ribose] polymerase) activation and diabetic endothelial dysfunction. Here, we tested whether PARP activity in circulating leukocytes was associated with coronary artery disease (CAD) among patients with type 2 diabetes mellitus (T2DM). Approach and Results: We performed observational and bidirectional Mendelian randomization studies of 3149 Chinese individuals with T2DM who underwent coronary angiography, with leukocyte PARP activity, 16 tag single-nucleotide polymorphisms in PARP1 and PARP2, and 17 CAD risk single-nucleotide polymorphisms analyzed. Of 3149 participants, 1180 who further received percutaneous coronary intervention were prospectively followed for 1 year to track major adverse cardiovascular and cerebrovascular events. Overall, greater PARP activity was cross-sectionally associated with an odds ratio of 1.23 for obstructive CAD, and prospectively with a hazard ratio of 1.34 for 1-year major adverse cardiovascular and cerebrovascular events after percutaneous coronary intervention (both P<0.001). Using a genetic score of 5 screened single-nucleotide polymorphisms in PARP1 and PARP2 as the instrumental variable, genetically predicted elevation in PARP activity showed a causal association with obstructive CAD (odds ratio=1.35, P<0.001). In contrast, the genetic risk of CAD had no significant effect on PARP activity. Ex vivo and in vitro cultures of human monocytes showed that rs747657, as the lead single-nucleotide polymorphism strongly associated with PARP activity, caused the differential binding of transcription factor GATA2 (GATA-binding protein 2) to an intronic regulatory region in PARP1, thus modulating PARP1 expression and PARP activity. Greater PARP activity may have causal roles in the development of obstructive CAD among patients with diabetes mellitus.

Kinase Phosphorylation-based Mechanisms of PARP Inhibitor Resistance During Synthetic Lethal Oncotherapy

Background: Poly-(ADP-Ribose) Polymerase (PARP) plays a central role in recovery from single-strand DNA (ssDNA) damage via base excision repair. When PARP activity is inhibited by a NAD+ mimetic analog, ssDNA is converted into a Double-Strand Break (DSB) during the S-phase in a cell cycle. However, the DSB site is repaired in a process of Homologous Recombination (HR) that is derived by genes such as BRCA1/2, PALB2, and RAD51. Under conditions of HR dysfunction, including mutations of BRCA1/2 (called BRCAness), PARP inhibitor (PARPi) induces “synthetic lethality” in BRCAness-specific cancer cells. Indeed, clinical trials using forms of PARPi that include olaparib, veliparib and rucaparib, have revealed that PARP inhibition produces a dramatic effect that actually arrests cancer progression. Its clinical efficiency is limited, however, due to the acquisition of PARPi resistance during long-term use of this inhibitor. Thus, it is important to elucidate the mechanisms of PARPi resistance. Methods: We searched the scientific literature published in PubMed, with a special focus on kinase phosphorylation that is involved in acquiring PARPi resistance. We also summarized the possible molecular events for recovering HR system, a key event for acquiring PARPi resistance. Results: CDK1 is a critical kinase for 5’-3’ DNA end resection, which is important for generating ssDNA for recruiting HR-priming factors. CDK12 is necessary for the transcription of HR-driver genes, such as BRCA1, BRCA2, RAD51 and ATR via the phosphorylation of RNA Pol-II. PLK-1 participates in driving HR via the phosphorylation of RAD51. The PI3K-AKT-mTOR signaling cascade is involved in BRCA1 induction via an ETS1 transcriptional pathway. Even under ATMdeficient conditions, the ATR-CHK1 axis compensates for loss in the DNA damage response, which results in HR recovery. The HGF receptor Met tyrosine kinase is responsible for promoting DNA repair by activating the PARP catalytic domain. Conclusion: These kinase-based signaling pathways are biologically important for understanding the compensatory system of HR, whereas inactivation of these kinases has shown promise for the release of PARPi resistance. Several lines of preclinical studies have demonstrated the potential use of kinase inhibitors to enhance PARPi sensitivity. We emphasize the clinical importance of chemical inhibitors as adjuvant drugs to block critical kinase activities and prevent the possible PARPi resistance.

PARP1 Impedes SIRT1-Mediated Autophagy during Degeneration of the Retinal Pigment Epithelium under Oxidative Stress.

The molecular mechanism underlying autophagy impairment in the retinal pigment epithelium (RPE) in dry age-related macular degeneration (AMD) is not yet clear. Based on the causative role of poly(ADP-ribose) polymerase 1 (PARP1) in RPE necrosis, this study examined whether PARP1 is involved in the autophagy impairment observed during dry AMD pathogenesis. We found that autophagy was downregulated following H2O2-induced PARP1 activation in ARPE-19 cells and olaparib, PARP1 inhibitor, preserved the autophagy process upon H2O2 exposure in ARPE-19 cells. These findings imply that PARP1 participates in the autophagy impairment upon oxidative stress in ARPE-19 cells. Furthermore, PARP1 inhibited autolysosome formation but did not affect autophagosome formation in H2O2-exposed ARPE-19 cells, demonstrating that PARP1 is responsible for impairment of late-stage autophagy in particular. Because PARP1 consumes NAD+ while exerting its catalytic activity, we investigated whether PARP1 impedes autophagy mediated by sirtuin1 (SIRT1), which uses NAD+ as its cofactor. A NAD+ precursor restored autophagy and protected mitochondria in ARPE-19 cells by preserving SIRT1 activity upon H2O2. Moreover, olaparib failed to restore autophagy in SIRT1-depleted ARPE-19 cells, indicating that PARP1 inhibits autophagy through SIRT1 inhibition. Next, we further examined whether PARP1-induced autophagy impairment occurs in the retinas of dry AMD model mice. Histological analyses revealed that olaparib treatment protected mouse retinas against sodium iodate (SI) insult, but not in retinas cotreated with SI and wortmannin, an autophagy inhibitor. Collectively, our data demonstrate that PARP1-dependent inhibition of SIRT1 activity impedes autophagic survival of RPE cells, leading to retinal degeneration during dry AMD pathogenesis.

Poly (ADP-ribose) polymerase inhibitor 3-methoxybenzamide enhances in vitro plant growth, microtuberization, and transformation efficiency of blue potato (Solanum tuberosum L. subsp. andigenum)

Poly(ADP-ribose) polymerases (PARP) are involved in the regulation of plant growth and development, especially under stress conditions. These enzymes post-translationally modify target proteins by adding ADP-ribose polymers. This study presents the effect of PARP chemical inhibitor 3-methoxybenzamide (3 MB) on in vitro plant growth, microtuber formation, and Agrobacterium-mediated transformation of blue potato (Solanum tuberosum L. subsp. andigenum). The addition of 3 MB (0.2 to 0.6 mM) significantly increased the growth and microtuber formation of in vitro propagated plants. The highest gain in plant fresh weight, root mass, and microtuberization was observed in clonal propagation medium with 0.4 mM 3 MB. Transformation frequency of blue potato internodes increased to 43%, 2.7-fold over the non-treated control (16%), when 0.2 mM 3 MB was included in the culture medium during the whole process. The addition of 3 MB during Agrobacterium transformation did not affect transgene copy number in regenerated plants. Southern blot analyses and histochemical staining for s-glucuronidase activity confirmed the presence and expression, respectively, of the uidA transgene in plant tissues. From these results, it can be concluded that the inhibition of PARP activity can increase biomass production and transformation rates for genetic improvement of blue potato.

Abstract A75: Serine auxotrophy: A novel metabolic vulnerability of platinum-resistant ovarian cancer?

Abstract Ovarian cancer accounts for the highest number of gynecologic-associated deaths in the developed world, and resistance to platinum-based therapy represents a major clinical and societal challenge in patients’ management. Since metabolism is intertwined with signaling pathways controlling cell death, we aimed to investigate to what extent metabolic adaptations could contribute to the development of the resistant phenotype. By performing isotope-labeled 13C-glucose tracer analysis in vitro, we found that, when they become resistant to platinum, ovarian cancer cells stop to synthetize serine and are characterized by significantly lower intracellular levels of this amino acid compared to sensitive cells. However, serine is required for cellular growth and survival, and resistant cells increase its uptake from the medium, becoming exquisitely vulnerable to serine starvation. We showed that, although resistant cells accumulate DNA damage upon platinum treatment, they have a higher capacity of repairing it because of increased poly(ADP)-ribose polymerase (PARP) activity, compared to the sensitive ones. Since PARP enzymes are major oxidized nicotinamide adenine dinucleotide (NAD+)-consuming enzymes, we collected evidence that serine synthesis downregulation, as a consequence of central carbon metabolic reshuffling, provides resistant cells with the advantage of sparing NAD+, thus sustaining PARP activation and allowing a more efficient DNA repair. We confirmed that downregulation of serine synthesis is a peculiar trait of resistant tumors also in vivo, using patient-derived xenografts (PDX) ovarian cancer models subjected to serine/glycine free diet. Moreover, analysis of The Cancer Genome Atlas Consortium (TCGA) ovarian cancer dataset revealed that tumors from platinum-resistant patients are characterized by a downregulation of serine biosynthetic enzymes, suggesting that serine auxotrophy could represent a novel and exploitable vulnerability of platinum-resistant ovarian cancers. Citation Format: Tom Van Nyen, Joao A.G. Duarte, Matteo Rossi, Mélanie Planque, Esther Zaal, Ali Talebi, Stijn Moens, Guy Eelen, Hugo Horlings, Johan Swinnen, Celia Berkers, Peter Carmeliet, Reuven Agami, Sarah-Maria Fendt, Diether Lambrechts, Daniela Annibali, Frederic Amant. Serine auxotrophy: A novel metabolic vulnerability of platinum-resistant ovarian cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A75.

TALAPRO-2: a placebo-controlled phase III study of talazoparib (TALA) plus enzalutamide (ENZA) for patients with first-line metastatic castration-resistant prostate cancer (mCRPC).

TPS5598 Background: TALA blocks poly(ADP-ribose) polymerase (PARP) activity and traps PARP on single-strand DNA breaks, preventing DNA damage repair (DDR) and causing death of cells with DDR alterations (eg BRCA1/2).a TALA has been approved in multiple countries as monotherapy for germline BRCA1/2-mutated human epidermal growth factor receptor 2-negative advanced breast cancer. ENZA is an androgen receptor (AR) inhibitor and an established therapy for mCRPC. As PARP activity has been shown to support AR function, inhibition of PARP is expected to reduce AR signaling and increase sensitivity to AR-directed therapies. In addition, AR blockade downregulates homologous recombination repair gene transcription which induces ‘ BRCAness ’. Therefore, combining TALA with ENZA in mCRPC may be efficacious regardless of DDR alterations. TALAPRO-2 (NCT03395197) is a Phase III, 2-part study to evaluate efficacy, safety, pharmacokinetics, and patient-reported outcomes (PROs) of TALA combined with ENZA. Methods: Enrollment goal is 1037 patients (19 patients, part 1 dose-finding; 1,018 patients, part 2 placebo-controlled). Key eligibility criteria: age ≥18 years; asymptomatic/mildly symptomatic mCRPC; ECOG performance status ≤1; metastatic disease (no brain metastases); and no prior life-prolonging systemic therapy for nonmetastatic CRPC or mCRPC. Prior therapies (excluding novel AR inhibitors) in the castration-sensitive (CSPC) setting are allowed. ADT must continue throughout the study. The randomized double-blind portion (part 2) will evaluate safety, efficacy, and PROs of TALA (0.5 mg QD) + ENZA (160 mg QD) vs placebo + ENZA (160 mg QD). Patients are stratified by prior novel hormonal therapy or docetaxel for CSPC (yes or no) and DDR alteration status (deficient vs nondeficient/unknown). The primary endpoint is radiographic progression-free survival (rPFS), defined as time to progression in soft tissue per RECIST v.1.1 or in bone per PCWG3 criteria by independent review or death. The key secondary endpoint is overall survival. Efficacy will be assessed radiographically every 8 weeks up to Week 25 and every 8–12 weeks thereafter. rPFS will be compared between the two arms by a 1-sided stratified log-rank test. Patient recruitment is ongoing in multiple regions including US, Europe/Eastern Europe, Israel, South America, South Africa, and Asia-Pacific region. aDDR alterations are defined as known/likely pathogenic variants or homozygous deletions. Funding: Pfizer Inc. Clinical trial information: NCT03395197 .

Poly (ADP-Ribose) polymerase-1 (PARP-1) overactivity plays a pathogenic role in bile acids-induced nephrotoxicity in cholestatic rats

Cholestatic liver disease is a clinical complication with a wide range of etiologies. The liver is the primary organ influenced by cholestasis. Other organs, rather than the liver (e.g., kidneys), could also be affected by cholestatic liver disease. Cholestasis-induced renal injury is known as cholemic nephropathy (CN). Although the structural and functional alterations of the kidney in cholestasis have been well described, the cellular and molecular mechanisms of CN are not well understood. Some studies mentioned the role of oxidative stress and mitochondrial impairment in CN. Several cellular targets, including proteins, lipids, and DNA, could be affected by oxidative stress. Poly (ADP-Ribose) polymerase-1 (PARP-1) is an enzyme that its physiological activity plays a fundamental role in DNA repair. However, PARP-1 overexpression is associated with enhanced oxidative stress and cell death. The current study was designed to evaluate the role of PARP-1 activity in the pathogenesis of CN. Bile duct ligated (BDL) rats were treated with nicotinamide (NA) as a PARP-1 inhibitor. Kidney, urine, and plasma samples were collected at scheduled time intervals (3, 7, 14, and 28 days after BDL surgery). Serum and urine biomarkers of kidney injury, markers of oxidative stress and DNA damage, PARP-1 expression and activity in the kidney tissue, inflammatory response, renal fibrosis markers, and kidney histopathological alterations were assessed. Significant changes in the serum and urine biomarkers of kidney injury were evident in the BDL rats. Markers of oxidative stress were increased, and tissue ATP levels and antioxidant capacity were decreased in the kidney of cholestatic animals. A significant increase in PARP-1 expression and activity was evident in BDL rats (3, 7, 14, and 28 days after BDL). Moreover, inflammatory response (IL-1β and TNF-α expression; and myeloperoxidase activity), renal tissue histopathological alterations, and kidney fibrosis (α-SMA and TGF-β expression, as well as collagen deposition) were detected in cholestatic animals. It was found that the PARP-1 inhibitor, NA (50 and 100 mg/kg, i.p), significantly mitigated cholestasis-induced renal injury. The positive effects of NA were more significant at a lower dose and the early stage of CN. These data indicate a pathogenic role for PARP-1 overexpression in CN.

Olaparib-Based Photoaffinity Probes for PARP-1 Detection in Living Cells.

The poly‐ADP‐ribose polymerase (PARP) is a protein from the family of ADP‐ribosyltransferases that catalyzes polyadenosine diphosphate ribose (ADPR) formation in order to attract the DNA repair machinery to sites of DNA damage. The inhibition of PARP activity by olaparib can cause cell death, which is of clinical relevance in some tumor types. This demonstrates that quantification of PARP activity in the context of living cells is of great importance. In this work, we present the design, synthesis and biological evaluation of photo‐activatable affinity probes inspired by the olaparib molecule that are equipped with a diazirine for covalent attachment upon activation by UV light and a ligation handle for the addition of a reporter group of choice. SDS‐PAGE, western blotting and label‐free LC‐MS/MS quantification analysis show that the probes target the PARP‐1 protein and are selectively outcompeted by olaparib; this suggests that they bind in the same enzymatic pocket. Proteomics data are available via ProteomeXchange with identifier PXD018661.

Novel Poly(ADP-ribose) Polymerase-1 Inhibitor DDHCB Inhibits Proliferation of BRCA Mutant Breast Cancer Cell In Vitro and In Vivo through a Synthetic Lethal Mechanism.

Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors are drugs that are effectively used to treat breast cancer. We synthesized a novel bromophenol derivative ethyl (E)-4-(2-(2,3-dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamido)benzoate (DDHCB) as a novel PARP-1 inhibitor. Our study found that DDHCB could inhibit PARP-1 activity with an IC50 value of 58.3 nM. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT) assay indicated that DDHCB could selectively inhibit proliferation of BRCA mutant cells and demonstrate the ability of synthetic lethality. DDHCB could also induce DNA double-strand breaks with the ability to increase the foci quantitation of γ-H2AX. Moreover, DDHCB could increase PARP-1-DNA trapping and inhibit PAR formation in HCC-1937 cells. Further investigation showed that DDHCB induced apoptosis and G2/M cycle arrest. Finally, we found that DDHCB inhibited the growth of HCC-1937 xenografts with low toxicity. In vivo mechanisms showed that the level of γ-H2AX was increased in the DDHCB-treated tumors, indicating the PARP-1 inhibition ability of DDHCB in vivo. Our study results indicated that the future development of DDHCB for the treatment of breast cancer is promising.

Targeting Nuclear NAD+ Synthesis Inhibits DNA Repair, Impairs Metabolic Adaptation and Increases Chemosensitivity of U-2OS Osteosarcoma Cells.

Osteosarcoma (OS) is the most common bone tumor in children and adolescents. Modern OS treatment, based on the combination of neoadjuvant chemotherapy (cisplatin + doxorubicin + methotrexate) with subsequent surgical removal of the primary tumor and metastases, has dramatically improved overall survival of OS patients. However, further research is needed to identify new therapeutic targets. Here we report that expression level of the nuclear NAD synthesis enzyme, nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1), increases in U-2OS cells upon exposure to DNA damaging agents, suggesting the involvement of the enzyme in the DNA damage response. Moreover, genetic inactivation of NMNAT1 sensitizes U-2OS osteosarcoma cells to cisplatin, doxorubicin, or a combination of these two treatments. Increased cisplatin-induced cell death of NMNAT1−/− cells showed features of both apoptosis and necroptosis, as indicated by the protective effect of the caspase-3 inhibitor z-DEVD-FMK and the necroptosis inhibitor necrostatin-1. Activation of the DNA damage sensor enzyme poly(ADP-ribose) polymerase 1 (PARP1), a major consumer of NAD+ in the nucleus, was fully blocked by NMNAT1 inactivation, leading to increased DNA damage (phospho-H2AX foci). The PARP inhibitor, olaparib, sensitized wild type but not NMNAT1−/− cells to cisplatin-induced anti-clonogenic effects, suggesting that impaired PARP1 activity is important for chemosensitization. Cisplatin-induced cell death of NMNAT1−/− cells was also characterized by a marked drop in cellular ATP levels and impaired mitochondrial respiratory reserve capacity, highlighting the central role of compromised cellular bioenergetics in chemosensitization by NMNAT1 inactivation. Moreover, NMNAT1 cells also displayed markedly higher sensitivity to cisplatin when grown as spheroids in 3D culture. In summary, our work provides the first evidence that NMNAT1 is a promising therapeutic target for osteosarcoma and possibly other tumors as well.

Nicotinamide mononucleotide administration after sever hypoglycemia improves neuronal survival and cognitive function in rats

Hypoglycemia-induced brain injury is a potential complication of insulin therapy in diabetic patients. Severe hypoglycemia triggers a cascade of events in vulnerable neurons that may lead to neuronal death and cognitive impairment even after glucose normalization. Oxidative stress and the activation of poly (ADP-ribose) polymerase-1 (PARP-1) are key events in this cascade. The production of reactive oxygen species (ROS) induces DNA damage and the consequent PARP-1 activation, which depletes NAD+ and ATP, resulting in brain injury. One of the key precursors of NAD+ is nicotinamide mononucleotide (NMN), which is converted to NAD+ and reduces production of ROS. Here we investigated whether NMN could reduce brain injury after severe hypoglycemia. We used a rat model of insulin-induced severe hypoglycemia and injected NMN (500 mmg/kg, i.p., one week) following 30 min of severe hypoglycemia, at the time of glucose administration. One week after severe hypoglycemia, hippocampal long-term potentiation (LTP), an electrophysiogic assay of synaptic plasticity, was examined and neuronal damage was assessed by Hematoxylin-Eosin staining. ROS accumulation, PARP-1 activation, NAD+ and ATP levels in hippocampus were also measured. Cognitive function was assessed using the Morris water maze 6 weeks after severe hypoglycemia. The addition of NMN reduced neuron death by 83 ± 3% (P < 0.05) after severe hypoglycemia. The hippocampal LTP was significantly reduced by severe hypoglycemia but showed recovery in the NMN addition group. NMN treatment also attenuated the severe hypoglycemia-induced spatial learning and memory impairment. Mechanically, we showed that NMN administration decreased ROS accumulation, suppressed PARP-1 activation, and restored levels of NAD+ and ATP in hippocampus. All these protective effects were reversed by 3-acetylpyridine (3-AP), which generates inactive NAD+. In summary, NMN administration following severe hypoglycemia could ameliorate neuronal damage and cognitive impairment caused by severe hypoglycemia. These results suggest that NMN may be a promising therapeutic drug to prevent hypoglycemia-induced brain injury.

Effects of the Poly(ADP-Ribose) Polymerase Inhibitor Olaparib in Cerulein-Induced Pancreatitis

Activation of the constitutive nuclear and mitochondrial enzyme poly (ADP-ribose) polymerase (PARP) has been implicated in the pathogenesis of cell dysfunction, inflammation, and organ failure in various forms of critical illness. The objective of our study was to evaluate the efficacy and safety of the clinically approved PARP inhibitor olaparib in an experimental model of pancreatitis in vivo and in a pancreatic cell line subjected to oxidative stress in vitro. The preclinical studies were complemented with analysis of clinical samples to detect PARP activation in pancreatitis. Mice were subjected to cerulein-induced pancreatitis; circulating mediators and circulating organ injury markers; pancreatic myeloperoxidase and malondialdehyde levels were measured and histology of the pancreas was assessed. In human pancreatic duct epithelial cells (HPDE) subjected to oxidative stress, PARP activation was measured by PAR Western blotting and cell viability and DNA integrity were quantified. In clinical samples, PARP activation was assessed by PAR (the enzymatic product of PARP) immunohistochemistry. In male mice subjected to pancreatitis, olaparib (3 mg/kg i.p.) improved pancreatic function: it reduced pancreatic myeloperoxidase and malondialdehyde levels, attenuated the plasma amylase levels, and improved the histological picture of the pancreas. It also attenuated the plasma levels of pro-inflammatory mediators (TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-12, IP-10, KC) but not MCP-1, RANTES, or the anti-inflammatory cytokine IL-10. Finally, it prevented the slight, but significant increase in plasma blood urea nitrogen level, suggesting improved renal function. The protective effect of olaparib was also confirmed in female mice. In HPDE cells subjected to oxidative stress olaparib (1 μM) inhibited PARP activity, protected against the loss of cell viability, and prevented the loss of cellular NAD levels. Olaparib, at 1μM to 30 μM did not have any adverse effects on DNA integrity. In human pancreatic samples from patients who died of pancreatitis, increased accumulation of PAR was demonstrated. Olaparib improves organ function and tempers the hyperinflammatory response in pancreatitis. It also protects against pancreatic cell injury in vitro without adversely affecting DNA integrity. Repurposing and eventual clinical introduction of this clinically approved PARP inhibitor may be warranted for the experimental therapy of pancreatitis.

Real-time monitoring of PARP1-dependent PARylation by ATR-FTIR\xa0spectroscopy

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD+ substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3’ phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD+ and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.

Probing the Conformational Changes Associated with DNA Binding to PARP1.

Poly(ADP-ribose) polymerase 1 (PARP1) is an important first responder in the mechanism of DNA repair in eukaryotic cells. It is also a validated drug target, with four different PARP inhibitors (PARPi) approved for the treatment of BRCA-negative cancers. Despite past efforts, many aspects of PARPi are poorly understood, in particular their ability to trap PARP1 on chromatin and the relationships between their potencies, cellular toxicities, and trapping efficiencies. Because PARP trapping is widely believed to originate in allosteric coupling between DNA binding and the catalytic site, we further investigated the binding properties of PARP1 to a model for DNA with a double-strand break in the presence and absence of PARPi. Specifically, we have used sequential mixing stopped-flow spectroscopy to identify a slow conformational change that follows rapid DNA binding. Using a range of DNA concentrations and different mutants of PARP1 we demonstrate that this conformational change is one of the steps of the "monkey bar mechanism" that promotes DNA-dependent dissociation of DNA. This conformational change also corresponds to the previously identified conformational change associated with DNA-dependent activation of PARP1. Despite linking the conformational change associated with DNA binding and release to DNA activation, we find no evidence for PARPi perturbing this allosteric coupling.

Lipoic Acid Decreases the Expression of Poly ADP-Ribose Polymerase and Inhibits Apoptosis in Diabetic Rats.

PurposeTo study the effects of lipoic acid on poly ADP-ribose polymerase (PARP) expression and apoptosis in diabetic rats.Materials and MethodsSprague–Dawley rats (n=30) with high-fat diet- and streptozotocin-induced diabetes were randomly divided into two groups: diabetic model (DM) group and lipoic acid (LA) treatment group; another 10 rats were selected as normal controls (NC). The serum levels of 8-hydroxy-2ʹ-deoxyguanosine, nitrotyrosine, and 8-isoprostane; sciatic nerve cell apoptosis index; and PARP expression were detected in the rats, and morphological changes in the sciatic nerve were recorded.ResultsThe blood glucose level in the DM and LA groups was significantly higher than that of the NC group (P<0.01). Compared to the NC group, the DM group showed demyelinating changes to sciatic nerve fibers. PARP expression; serum levels of 8-hydroxy-2-deoxyguanosine, nitrotyrosine, and 8-isoprostane; and the apoptosis index of sciatic nerve cells were significantly higher than those of the NC group (P<0. 01). Following LA treatment, the above indices showed significant improvement (P<0.01).ConclusionLipoic acid may improve the symptoms of diabetic neuropathy by reducing PARP activity and inhibiting apoptosis.

Baicalein attenuates caspase-independent cells death via inhibiting PARP-1 activation and AIF nuclear translocation in cerebral ischemia/reperfusion rats.

It is reported that baicalein can activate PI3K/AKT pathway, inhibit caspase activation and reduce cerebral infarct volume in middle cerebral artery occlusion (MCAO) rats. However, a caspase-independent mechanism initiated by poly (ADP-ribose) polymerase-1 (PARP-1) activation has been reported to make more contribution to cells death after ischemic stroke. In the present study, we established a cerebral ischemia/reperfusion (I/R) rat model through middle cerebral artery occlusion following reperfusion to investigate the mechanisms of ischemic tissue recovery following baicalein treatment. The data showed that baicalein treatment at dose of 100 mg/kg for 7 days significantly inhibited the release of cytokines, activation of PARP-1, nuclear translocation of apoptosis-inducing factor (AIF) and macrophage migration inhibitory factor (MIF) in cerebral I/R rats, therefore decreased cerebral infarct volume and neurological scores. Then, we further investigated the signal transduction mechanisms of ischemic tissue protection by baicalein in vitro. Following oxygen and glucose deprivation (OGD) in SH-SY5Y cells, the mitochondrial AIF was translocated into nucleus after 12 h. The co-immunoprecipitation analysis showed that the interaction between AIF and MIF was activated by OGD and subsequently resulted in MIF nuclear translocation. Also, the baicalein inhibited apoptosis, reduced oxidative stress, protected mitochondrial function and restored mitochondrial membrane potential in OGD cells. The results obtained from both in vivo and in vitro study demonstrated the PARP-1/AIF pathway involved in mechanisms of baicalein to protect the cerebral tissues from ischemic injury.