Effect of poly(ADP-ribosyl)ation inhibitors on the genotoxic effects of the boron neutron capture reaction

The boron neutron capture (BNC) reaction results from the interaction of 10B with low-energy thermal neutrons and gives rise to highly damaging lithium and alpha-particles. In this work the genotoxicity caused by the BNC reaction in V79 Chinese hamster cells was evaluated in the presence of poly(ADP-ribosyl)ation inhibitors. Poly(ADP-ribose) polymerase-1 (PARP-1), the most important member of the PARP enzyme family, is considered to be a constitutive factor of the DNA damage surveillance network present in eukaryotic cells, acting through a DNA break sensor function. Inhibition of poly(ADP-ribosyl)ation was achieved with the classical compound 3-aminobenzamide (3-AB), and with two novel and very potent inhibitors, 5-aminoisoquinolinone (5-AIQ) and PJ-34. Dose-response increases in the frequencies of aberrant cells excluding gaps (%ACEG) and chromosomal aberrations excluding gaps per cell (CAEG/cell) were observed for increasing exposures to the BNC reaction. The presence of 3-AB did not increase the %ACEG or CAEG/cell, nor did it change the pattern of the induced chromosomal aberrations. Results with 5-AIQ and PJ-34 were in agreement with the results obtained with 3-AB. We further studied the combined effect of a PARP inhibitor and a DNA-dependent protein kinase (DNA-PK) inhibitors (3-AB and wortmannin, respectively) on the genotoxicity of the BNC reaction, by use of the cytokinesis-block micronucleus assay. DNA-PK is also activated by DNA breaks and binds DNA ends, playing a role of utmost importance in the repair of double-strand breaks. Our results show that the inhibition of poly(ADP-ribosyl)ation does not particularly modify the genotoxicity of the BNC reaction, and that PARP inhibition together with a concomitant inhibition of DNA-PK revealed barely the same sensitizing effect as DNA-PK inhibition per se.     

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent γ-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered γ-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.     

Partial protection by poly(ADP-ribose) polymerase inhibitors from nitroxyl-induced cytotoxity in thymocytes

Nitroxyl (NO⁻/HNO), has been proposed to be one of the NO^•-derived cytotoxic species. Although the biological effect of nitroxyl is largely unknown, it has been reported to cause DNA breakage and cytotoxicity. We have therefore investigated whether NO⁻/HNO-induced DNA single-strand breakage activates the nuclear nick sensor enzyme poly(ADP-ribose) polymerase (PARP) and whether PARP activation affects the mode of NO⁻/HNO- induced cell death. NO⁻/HNO generated from Angeli’s salt (AS, sodium trioxodinitrate) (0–300 μM) induced DNA single-strand breakage, PARP activation, and a concentration-dependent cytotoxicity in murine thymocytes. AS-induced cell death was also accompanied by decreased mitochondrial membrane potential and increased secondary superoxide production. The cytotoxicity of AS, as measured by propidium iodide uptake, was abolished by electron acceptors potassium ferricyanide, TEMPOL, the intracellular calcium chelator BAPTA-AM, and by PARP inhibitors 3-aminobenzamide (3-AB) and PJ-34. The cytoprotective effect of 3-AB was paralleled by increased output of AS-induced apoptotic parameters such as phosphatidylserine exposure, caspase activation, and DNA fragmentation. No significant increase in tyrosine nitration could be observed in AS-treated thymocytes as opposed to peroxynitrite-treated cells, indicating that tyrosine nitration is not likely to contribute to NO⁻/HNO-induced cytotoxicity. Our results demonstrate that NO⁻/HNO-induced PARP activation shifts the default apoptotic cell death toward necrosis in thymocytes. However, as total PARP inhibition resulted only in 30% cytoprotection, PARP-independent mechanisms dominate NO⁻/HNO-induced cytotoxicity in thymocytes.     

Pharmacological inhibition of poly(ADP-ribose) polymerase inhibits angiogenesis

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which plays an important role in regulating cell death and cellular responses to DNA repair. Pharmacological inhibitors of PARP are being considered as treatment for cancer both in monotherapy as well as in combination with chemotherapeutic agents and radiation, and were also reported to be protective against untoward effects exerted by certain anticancer drugs. Here we show that pharmacological inhibition of PARP with 3-aminobenzamide or PJ-34 dose-dependently reduces VEGF-induced proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro. These results suggest that treatment with PARP inhibitors may exert additional benefits in various cancers and retinopathies by decreasing angiogenesis.     

Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

Poly(ADP-ribose)polymerase (PARP) inhibitors decrease angiogenesis through reducing vascular endothelium growth factor (VEGF) induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). In contrast to VEGF, pigment epithelium-derived factor (PEDF) has been demonstrated to act as a strong endogenous inhibitor of angiogenesis. Here, we show that PARP inhibition with a specific inhibitor PJ-34 or specific PARP antisense oligonucleotide upregulates hyperglycemia-induced PEDF expression in HUVECs in a dose-dependent manner. This results in the retard of activation of p38 MAP kinase and the concomitant decrease in cell apoptosis. These results give the first direct demonstration that PEDF might represent a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent.     

Another “String to the Bow” of PJ34, a Potent Poly(ADP-Ribose)Polymerase Inhibitor: An Antiplatelet Effect through P2Y12 Antagonism?

Background

Neuro- and vasoprotective effects of poly(ADP-ribose)polymerase (PARP) inhibition have been largely documented in models of cerebral ischemia, particularly with the potent PARP inhibitor PJ34. Furthermore, after ischemic stroke, physicians are faced with incomplete tissue reperfusion and reocclusion, in which platelet activation/aggregation plays a key role. Data suggest that certain PARP inhibitors could act as antiplatelet agents. In that context, the present in vitro study investigated on human blood the potential antiplatelet effect of PJ34 and two structurally different PARP inhibitors, DPQ and INO-1001.

Methods and results

ADP concentrations were chosen to induce a biphasic aggregation curve resulting from the successive activation of both its receptors P2Y₁ and P2Y₁₂. In these experimental conditions, PJ34 inhibited the second phase of aggregation; this effect was reduced by incremental ADP concentrations. In addition, in line with a P2Y₁₂ pathway inhibitory effect, PJ34 inhibited the dephosphorylation of the vasodilator stimulated phosphoprotein (VASP) in a concentration-dependent manner. Besides, PJ34 had no effect on platelet aggregation induced by collagen or PAR1 activating peptide, used at concentrations inducing a strong activation independent on secreted ADP. By contrast, DPQ and INO-1001 were devoid of any effect whatever the platelet agonist used.

Conclusions

We showed that, in addition to its already demonstrated beneficial effects in in vivo models of cerebral ischemia, the potent PARP inhibitor PJ34 exerts in vitro an antiplatelet effect. Moreover, this is the first study to report that PJ34 could act via a competitive P2Y₁₂ antagonism. Thus, this antiplatelet effect could improve post-stroke reperfusion and/or prevent reocclusion, which reinforces the interest of this drug for stroke treatment.     

Poly (ADP-ribose) polymerase plays an important role in intermittent hypoxia-induced cell death in rat cerebellar granule cells

Background

Episodic cessation of airflow during sleep in patients with sleep apnea syndrome results in intermittent hypoxia (IH). Our aim was to investigate the effects of IH on cerebellar granule cells and to identify the mechanism of IH-induced cell death.

Methods

Cerebellar granule cells were freshly prepared from neonatal Sprague-Dawley rats. IH was created by culturing the cerebellar granule cells in the incubators with oscillating O₂ concentration at 20% and 5% every 30 min for 1-4 days. The results of this study are based on image analysis using a confocal microscope and associated software. Cellular oxidative stress increased with increase in IH. In addition, the occurrence of cell death (apoptosis and necrosis) increased as the duration of IH increased, but decreased in the presence of an iron chelator (phenanthroline) or poly (ADP-ribose) polymerase (PARP) inhibitors [3-aminobenzamide (3-AB) and DPQ]. The fluorescence of caspase-3 remained the same regardless of the duration of IH, and Western blots did not detect activation of caspase-3. However, IH increased the ratio of apoptosis-inducing factor (AIF) translocation to the nucleus, while PARP inhibitors (3-AB) reduced this ratio.

Results

According to our findings, IH increased oxidative stress and subsequently leading to cell death. This effect was at least partially mediated by PARP activation, resulting in ATP depletion, calpain activation leading to AIF translocation to the nucleus.

Conclusions

We suggest that IH induces cell death in rat primary cerebellar granule cells by stimulating oxidative stress PARP-mediated calpain and AIF activation.     

Poly(ADP-ribose) polymerase inhibitor PJ-34 reduces mesenteric vascular injury induced by experimental cardiopulmonary bypass with cardiac arrest

The aim of this study was to investigate effects of poly(ADP-ribose) polymerase (PARP) inhibition on mesenteric vascular function and metabolism in an experimental model of cardiopulmonary bypass (CPB) with cardiac arrest. Twelve anesthetized dogs underwent 90-min hypothermic CPB. After 60 min of cardiac arrest, reperfusion was started for 40 min following application of either saline vehicle (control, n = 6) or a potent PARP inhibitor, PJ-34 (10 mg/kg iv bolus and 0.5 mg.kg(-1).min(-1) infusion for 20 min, n = 6). PJ-34 led to better recovery of cardiac output (2.2 +/- 0.1 vs. 1.8 +/- 0.2 l/min in control) and mesenteric blood flow (175 +/- 38 vs. 83 +/- 4 ml/min, P < 0.05 vs. control) after reperfusion. The impaired vasodilator response of the superior mesenteric artery to acetylcholine, assessed in the control group after CPB (-32.8 +/- 3.3 vs. -57.6 +/- 6.6% at baseline, P < 0.05), was improved by PJ-34 (-50.3 +/- 3.6 vs. -54.3 +/- 4.1% at baseline, P < 0.05 vs. control). Although plasma nitrate/nitrite concentrations were not significantly different between groups, mesenteric nitric oxide synthase activity was increased in the PJ-34 group (P < 0.05). Moreover, the treated group showed a marked attenuation of mesenteric venous plasma myeloperoxidase levels after CPB compared with the control group (75 +/- 1 vs. 135 +/- 9 ng/ml, P < 0.05). Pharmacological PARP inhibition protects against development of post-CPB mesenteric vascular dysfunction by improving hemodynamics, restoring nitric oxide production, and reducing neutrophil adhesion.     

Blockade of PARP activity attenuates poly(ADP-ribosyl)ation but offers only partial neuroprotection against NMDA-induced cell death in the rat retina

Recent reports have linked neuronal cell death by necrosis to poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation. It is believed that under stress, the activity of this enzyme is up-regulated, resulting in extensive poly(ADP-ribosyl)ation of nuclear proteins, using NAD(+) as its substrate, which, in turn, leads to the depletion of NAD(+). In efforts to restore the level of NAD(+), depletion of ATP occurs, resulting in the shutdown of ATP-dependent ionic pumps. This results in cell swelling and eventual loss of membrane selectivity, hallmarks of necrosis. Reports from in vitro and in vivo studies in the brain have shown that NMDA receptor activation stimulates PARP activity and that blockade of the enzyme provides substantial neuroprotection. The present study was undertaken to determine whether PARP activity is regulated by NMDA in the rat retina, and whether blockade of PARP activity provides protection against toxic effects of NMDA. Rat retinas exposed to intravitreal injections containing NMDA, with or without the PARP inhibitor N-(6-oxo-5, 6-dihydrophenanthridin-2-yl)-(N,-dimethylamino) acetamide hydrochloride (PJ-34), were assessed for changes in PARP-1 activity as evidenced by poly(ADP-ribosyl)ation (PAR), loss of membrane integrity, morphological indicators of apoptosis and necrosis, and ganglion cell loss. Results showed that: NMDA increased PAR formation in a concentration-dependent manner and caused a decline in retinal ATP levels; PJ-34 blockade attenuated the NMDA-induced formation of PAR and decline in ATP; NMDA induced the loss of membrane selectivity to ethidium bromide (EtBr) in inner retinal neurons, but loss of membrane selectivity was not prevented by blocking PARP activity; cells stained with EtBr, or reacted for TUNEL-labeling, displayed features characteristic of both apoptosis and necrosis. In the presence of PJ-34, greater numbers of cells exhibited apoptotic features; PJ-34 provided partial neuroprotection against NMDA-induced ganglion cell loss. These findings suggest that although blockade of PARP activity fully attenuates NMDA-induced PAR formation and loss of retinal ATP content, and improves the survival of select populations of ganglion cells, this approach does not provide full neuroprotection. In contrast, blockade of PARP activity promotes apoptotic-like cell death in the majority of cells undergoing cell death. Furthermore, these studies show that the loss of membrane selectivity is not dependent upon PAR formation or the resulting decline of ATP, and suggests that an alternative pathway, other than PARP activation, exists to mediate this event.     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

Cytochrome c release upon Fas receptor activation depends on translocation of full-length bid and the induction of the mitochondrial permeability transition.

In Jurkat cells Bid was cleaved upon activation of the Fas receptor with an anti-Fas antibody. The caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-CH(2)F (IETD) prevented the cleavage of Bid and the loss of viability. The nuclear enzyme poly(ADP-ribose)polymerase (PARP) was also cleaved upon the activation of caspases, and IETD similarly prevented PARP cleavage. The PARP inhibitor 3-aminobenzamide (3-AB) restored the cell killing in the presence of IETD, an effect that occurred without restoration of the cleavage of Bid or PARP. In the presence of 3-AB and IETD, translocation occurred of full-length Bid to the mitochondria. The induction of the mitochondrial permeability transition (MPT) was documented by the cyclosporin A (CyA) sensitivity of the release of cytochrome c, the release of malate dehydrogenase from the mitochondrial matrix, the loss of the mitochondrial membrane potential, and the pronounced swelling of these organelles, as assessed by electron microscopy. In addition to preventing all evidence of the MPT, CyA prevented the loss of cell viability, without effect on the cleavage of either Bid or PARP. The prevention of PARP cleavage by inhibition of caspase-3 resulted in a 10-fold activation of the enzyme and a resultant depletion of NAD and ATP. The PARP inhibitor 3-AB prevented the loss of NAD and ATP. Depletion of ATP by metabolic inhibitors similarly prevented the cell killing. It is concluded that the cleaving of PARP in Fas-mediated apoptosis allowed expression of an energy-dependent cell death program that included the translocation of full-length Bid to the mitochondria with induction of the MPT.     

Beneficial effects of poly (ADP-ribose) polymerase inhibition against the reperfusion injury in heart transplantation

We investigated the effect of 3-aminobenzamide (3-AB), an inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), against early ischemia/reperfusion (IR) injury in heart transplantation. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (60 min). In these conditions, and in the absence of 3-AB treatment, clear signs of oxidative stress, such as lipid peroxidation, increase in protein carbonyls and DNA strand breaks, were evident; PARP was markedly activated in concomitance with a significant NAD + and ATP depletion. The results of microscopic observations (nuclear clearings, plasma membrane discontinuity), and the observed rise in the serum levels of heart damage markers, suggested the development of necrotic processes while, conversely, no typical sign of apoptosis was evident. Compared to the effects observed in untreated IR heart, the administration of 3-AB (10 mg/kg to the donor and to the recipient animal), but not that of its inactive analogue 3-aminobenzoic acid, significantly modified the above parameters: the levels of oxidative stress markers were significantly reduced; PARP activation was markedly inhibited and this matched a significant rise in NAD + and ATP levels. PARP inhibition also caused a reduced release of the cardiospecific damage markers and attenuated morphological cardiomyocyte alterations, save that, in this condition, we noted the appearance of typical apoptotic markers: activation of caspase-3, oligonucleosomal DNA fragmentation, ISEL positive nuclei. Possible mechanisms for these effects are discussed, in any case the present results indicate that PARP inhibition has an overall beneficial effect against myocardial reperfusion injury, mainly due to prevention of energy depletion. In this context, the signs of apoptosis observed under 3-AB treatment might be ascribed to the maintenance of sufficient intracellular energy levels. These latter allow irreversible damages triggered during the ischemic phase to proceed towards apoptosis instead of towards necrosis, as it appears to happen when the energetic pools are depleted by high PARP activity.     

Beneficial Effects of Poly (ADP-ribose) Polymerase Inhibition Against the Reperfusion Injury in Heart Transplantation

We investigated the effect of 3-aminobenzamide (3-AB), an inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), against early ischemia/reperfusion (IR) injury in heart transplantation. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (60 min). In these conditions, and in the absence of 3-AB treatment, clear signs of oxidative stress, such as lipid peroxidation, increase in protein carbonyls and DNA strand breaks, were evident; PARP was markedly activated in concomitance with a significant NAD + and ATP depletion. The results of microscopic observations (nuclear clearings, plasma membrane discontinuity), and the observed rise in the serum levels of heart damage markers, suggested the development of necrotic processes while, conversely, no typical sign of apoptosis was evident. Compared to the effects observed in untreated IR heart, the administration of 3-AB (10 mg/kg to the donor and to the recipient animal), but not that of its inactive analogue 3-aminobenzoic acid, significantly modified the above parameters: the levels of oxidative stress markers were significantly reduced; PARP activation was markedly inhibited and this matched a significant rise in NAD + and ATP levels. PARP inhibition also caused a reduced release of the cardiospecific damage markers and attenuated morphological cardiomyocyte alterations, save that, in this condition, we noted the appearance of typical apoptotic markers: activation of caspase-3, oligonucleosomal DNA fragmentation, ISEL positive nuclei. Possible mechanisms for these effects are discussed, in any case the present results indicate that PARP inhibition has an overall beneficial effect against myocardial reperfusion injury, mainly due to prevention of energy depletion. In this context, the signs of apoptosis observed under 3-AB treatment might be ascribed to the maintenance of sufficient intracellular energy levels. These latter allow irreversible damages triggered during the ischemic phase to proceed towards apoptosis instead of towards necrosis, as it appears to happen when the energetic pools are depleted by high PARP activity.     

Effect of DNA repair inhibitor (3-aminobenzamide) on genetic stability of loach (Misgurnus fossilis) embryos derived from cryopreserved sperm

Semen cryopreservation is widely used in clinical medicine, agriculture, aquaculture and biomedical research, but it is an inefficient technique that induces extensive cytoplasmic damage and loss of fertilising ability. Whether any genetic damage (i.e. DNA strand breakage or mutation) is also induced is still unclear. However, previous data has indicated that this is likely. The present study was designed to explore this possibility further by using inhibitors of the DNA repair system to block DNA repair in embryos derived from cryopreserved spermatozoa. If cryopreservation causes strand breaks in sperm DNA it might be expected that inhibition of a repair enzyme such as poly(ADP-ribose) polymerase (PARP) would enhance any such negative effect of cryopreservation. To check this hypothesis 3-aminobenzamide (3-AB) was used as an inhibitor of PARP. Weather loach (Misgurnus fossilis) eggs were fertilised using cryopreserved as well as fresh spermatozoa. Embryos derived from cryopreserved spermatozoa were exposed to 10 mM 3-AB for 2 h after fertilisation. The experiments were carried out using 43,544 embryos from 5 females and 10 males. Embryo survival was evaluated at different stages until the hatching stage. Sperm cryopreservation significantly decreased embryo survival (53.6+/-2.79% compared to 76.97+/-2.79% of control; P<0.01). The addition of 3-AB to the medium with embryos derived from cryopreserved sperm further decreased embryo survival from 53.6+/-2.79% to 46.1+/-2.79% (P<0.01) whereas there was no adverse effect of 3-AB exposed embryos derived from fresh sperm (76.97+/-2.79% of control compared to 74.8+/-2.79% of control+3-AB). The effect of 3-AB provides indirect evidence that cryopreservation might induce instability in sperm DNA, and that such damage can be repaired by the oocyte repair system after fertilisation.     

Effects of poly(ADP-ribose) polymerase inhibition on dysfunction of non-adrenergic non-cholinergic neurotransmission in gastric fundus in diabetic rats

Diabetes mellitus compromises nitric oxide (NO)-mediated endothelium-dependent relaxation of blood vessels, which has been linked to the excessive generation of reactive oxygen species. There are also deleterious effect on nitrergic innervation, contributing to autonomic neuropathy symptoms such as impotence and gastroporesis. Poly(ADP-ribose) polymerase (PARP) is a nuclear protein stimulated by DNA damage, caused, for example, by oxidative stress. Activation has been linked to impaired endothelial nitric oxide synthase (eNOS)-mediated vasodilation in experimental diabetes. There is no information on the potential role of PARP in nitrergic nerve dysfunction, therefore, the aim was to examine the effects of PARP inhibition, using 3-aminobenzamide (3-AB) on neurally mediated gastric fundus relaxation in streptozotocin-induced diabetic rats. Eight weeks of diabetes caused a 42.5% deficit in maximum relaxation of in vitro gastric fundus strips to electrical stimulation of the non-adrenergic non-cholinergic innervation. This was largely prevented or corrected (4 weeks of treatment following 4 weeks of untreated diabetes) by 3-AB. Diabetes also markedly attenuated the maintenance of relaxation responses to prolonged stimulation, and this was partially corrected by 3-AB treatment. Experiments in the presence of the NOS inhibitor, N(G)-nitro-L-arginine, and/or blockade of the co-transmitter, vasoactive intestinal polypeptide, by alpha-chymotrypsin, showed that the beneficial effects of 3-AB were primarily due to improved nitrergic neurotransmission. Thus, PARP plays an important role in defective nitrergic neurotransmission in experimental diabetes, which may have therapeutic implications for treatment of aspects of diabetic autonomic neuropathy.     

One-pot tandem Hurtley–retro-Claisen–cyclisation reactions in the synthesis of 3-substituted analogues of 5-aminoisoquinolin-1-one (5-AIQ), a water-soluble inhibitor of PARPs

Poly(ADP-ribose)polymerase-1 (PARP-1) is an important target for drug design for several therapeutic applications. 5-Aminoisoquinolin-1-one (5-AIQ) is a highly water-soluble lead compound; synthetic routes to 3-substituted analogues were explored. Tandem Hurtley coupling of β-diketones with 2-bromo-3-nitrobenzoic acid, retro-Claisen acyl cleavage and cyclisation gave the corresponding 3-substituted 5-nitroisocoumarins. Treatment with ammonia at high temperature and reduction with tin(II) chloride gave eleven target 3-substituted 5-AIQs, which were all soluble in water (>1% w/v) as their HCl salts. Most were more potent than 5-AIQ as inhibitors of PARP-1 and of PARP-2 in vitro, the most active being 5-amino-3-methylisoquinolin-1-one (PARP-1: IC₅₀ = 0.23 μM vs IC₅₀ = 1.6 μM for 5-AIQ). Some rationalisation of the SAR was achieved through molecular modelling.