Three-component Castagnoli-Cushman reaction with ammonium acetate delivers 2-unsubstituted isoquinol-1-ones as potent inhibitors of poly(ADP-ribose) polymerase (PARP)

An earlier described three-component variant of the Castagnoli-Cushman reaction employing homophthalic anhydrides, carbonyl compound and ammonium acetate was applied towards the preparation of 1-oxo-3,4-dihydroisoquinoline-4-carboxamides with variable substituent in position 3. These compounds displayed inhibitory activity towards poly(ADP-ribose) polymerase (PARP), a clinically validated cancer target. The most potent compound (PARP1/2 IC₅₀ = 22/4.0 nM) displayed the highest selectivity towards PARP2 in the series (selectivity index = 5.5), more advantageous ADME prameters compared to the clinically used PARP inhibitor Olaparib.     

Discovery of quinazolinone and quinoxaline derivatives as potent and selective poly(ADP-ribose) polymerase-1/2 inhibitors

Two classes of quinazolinone derivatives and quinoxaline derivatives were identified as potent and selective poly(ADP-ribose) polymerase-1 and 2 (PARP-1) and (PARP-2) inhibitors, respectively. In PARP enzyme assays using recombinant PARP-1 and PARP-2, quinazolinone derivatives displayed relatively high selectivity for PARP-1 and quinoxaline derivatives showed superior selectivity for PARP-2. SBDD analysis via a combination of X-ray structural study and homology modeling suggested distinct interactions of inhibitors with PARP-1 and PARP-2. These findings provide a new structural framework for the design of selective inhibitors for PARP-1 and PARP-2.     

Novel alkoxybenzamide inhibitors of poly(ADP-ribose) polymerase

We have previously described poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors based on a substituted benzyl-phthalazinone scaffold. As an alternative chemical template, a novel series of alkoxybenzamides were developed with restricted conformation through intramolecular hydrogen bond formation; the compounds exhibit low nM enzyme and cellular activity as PARP-1 inhibitors.     

Discovery of novel benzo[b][1,4]oxazin-3(4H)-ones as poly(ADP-ribose)polymerase inhibitors

Structure based drug design of a series of novel 1,4-benzoxazin-3-one derived PARP-1 inhibitors are described. The synthesis, enzymatic & cellular activities and pharmacodynamic effects are described. Optimized analogs demonstrated inhibition of poly-ADP-ribosylation in SW620 tumor bearing nude mice through 24 h following a single dose.     

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.     

Development and validation of high-throughput screening assays for poly(ADP-ribose) polymerase-2 inhibitors

Poly(ADP-ribose) polymerase-1 and -2 (PARP1/2) are two key facilitators of DNA repair and are implicated in the pathogenesis of cancers and several chronic diseases. Inhibitors of PARP1/2 have shown powerful therapeutic effects in the treatment of cancer, cerebral ischemia, and inflammation. In addition, evidence from several studies suggests unique functions for PARP2 in genome surveillance, spermatogenesis, adipogenesis, and T cell development, and PARP2-specific inhibitors might have many other applications. To acquire PARP1/2 inhibitors, many high-throughput screening (HTS) assays for PARP1 inhibitors have been developed. However, detailed screening assays for PARP2 inhibitors have not been reported. Herein, three HTS assays for PARP2 inhibitors were developed and validated with reference inhibitors in each case. The results suggest that the HTS assays for PARP2 inhibitors using chemical quantification of NAD⁺, biotin-based quantification of PAR, and ELISA quantification of PAR are sensitive, robust, and cost effective.     

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.     

Expression of human poly(ADP-ribose) polymerase in Saccharomyces cerevisiae

The coding sequence for human poly(ADP-ribose) polymerase was expressed inducibly in Saccharomyces cerevisiae from a low-copy-number plasmid vector. Cell free extracts of induced cells had poly(ADPribose) polymerase activity when assayed under standard conditions; activity could not be detected in non-induced cell extracts. Induced cells formed poly(ADP-ribose) in vivo, and levels of these polymers increased when cells were treated with the alkylating agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG). The cytotoxicity of this agent was increased in induced cells, and in vivo labelling with [³H]adenine further decreased their viability. Increased levels of poly(ADP-ribose) found in cells treated with the alkylating agent were not accompanied by lowering of the NAD concentration.     

Neuroprotective effects of ONO-1924H, an inhibitor of poly ADP-ribose polymerase (PARP), on cytotoxicity of PC12 cells and ischemic cerebral damage

N-[3-(4-Oxo-3,4-dihydro-phthalazin-1-yl)phenyl]-4-(morpholin-4-yl) butanamide methanesulfonate monohydrate (ONO-1924H) is a novel inhibitor of poly ADP-ribose polymerase (PARP). In this study, we examined the effects of ONO-1924H on cytotoxicity induced by hydrogen peroxide in PC12 cells in vitro and cerebral damage and neurological deficits induced by middle cerebral artery (MCA) thrombus occlusion in vivo in rat. In the in vitro cytotoxicity assay, exposure to 0.5 mmol/L hydrogen peroxide induced cell death in differentiated PC12 cells. ONO-1924H, a PARP inhibitor (Ki=0.21 micromol/L), reduced cell death in a concentration-dependent manner that was correlated with inhibition of PARP activation. A 50% reduction in cell death (EC50) was achieved with 2.4 micromol/L ONO-1924H. In the MCA occlusion model, ONO-1924H was injected intravenously at doses of 3, 10 and 30 mg/kg/h for 3 h, and cerebral damage and neurological deficits were estimated 24 h after MCA occlusion. ONO-1924H treatment led to a significant decrease in cerebral damage in the 10 mg/kg/h-treated group (P < 0.05) and the 30 mg/kg/h-treated group (P < 0.01). Further, ONO-1924H at doses of 30 mg/kg/h significantly (P < 0.05) improved neurological deficits. These findings suggest that the novel PARP inhibitor, ONO-1924H, affords effective neuroprotection and is a useful therapeutic candidate for the treatment of ischemic stroke.     

Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V_max) and the michaelis constant (k_m) of PARG reaction were 4.46 μM and 128.33 μmol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 μM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.     

Functional analysis of poly(ADP-ribose) polymerase in Drosophila melanogaster

Poly(ADP-ribose) polymerase (PARP) is conserved in eukaryotes. To analyze the function of PARP, we isolated and characterized the gene for PARP in Drosophila melanogaster. The PARP gene consisted of six translatable exons and spanned more than 50 kb. The DNA binding domain is encoded by exons 1-4. Although the consensus cleavage site of CED-3 like protease during apoptosis is conserved from human to Xenopus laevis PARPs, it is neither conserved in the corresponding region of Drosophila nor Sarcophaga peregrina. There are two cDNAs species in Drosophila. One cDNA could encode the full length PARP protein (PARP I), while the other is a truncated cDNA which could encode a partial-length PARP protein (PARP II), which lacks the automodification domain and is possibly produced by alternative splicing. The expression of these two forms of PARP in E. coli demonstrated that while PARP II has the catalytic NAD-binding domain and DNA-binding domain it is enzymatically inactive. On the other hand PARP I is active. A deletion mutant of PARP gene could grow to the end of embryogenesis but did not grow to the adult fly. These results suggest that the PARP gene plays an important function during the development of Drosophila.     

NAD+ repletion prevents PARP-1-induced glycolytic blockade and cell death in cultured mouse astrocytes

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that is involved in DNA repair and activated by DNA damage. When activated, PARP-1 consumes NAD(+) to form ADP-ribose polymers on acceptor proteins. Extensive activation of PARP-1 leads to glycolytic blockade, energy failure, and cell death. These events have been postulated to result from NAD(+) depletion. Here, we used primary astrocyte cultures to directly test this proposal, utilizing the endogenous expression of connexin-43 hemichannels by astrocytes to manipulate intracellular NAD(+) concentrations. Activation of PARP-1 with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) produced NAD(+) depletion, glycolytic blockade, and cell death. Cultures incubated in high (10mM) extracellular concentrations of NAD(+) after MNNG exposure showed normalization of intracellular NAD(+) concentrations. Repletion of intracellular NAD(+) in this manner completely restored glycolytic capacity and prevented cell death. These results suggest that NAD(+) depletion is the cause of glycolytic failure after PARP-1 activation.     

The inhibition of poly(ADP-ribose) polymerase enhances growth rates of ataxia telangiectasia cells

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which is activated in response to genotoxic insults by binding damaged DNA and attaching polymers of ADP-ribose to nuclear proteins at the expense of its substrate NAD+. In persons affected with ataxia telangiectasia (A-T), associated mutations in the ataxia telangiectasia mutated gene render cells unable to cope with the genotoxic stresses from ionizing radiation and oxidative damage, thus resulting in a higher concentration of unrepaired DNA damage and the activation of PARP in an uncontrolled manner. In primary A-T fibroblasts, we observed a 58-96% increase in PARP activity and a concomitant loss of cellular NAD+ and ATP content. PARP protein by Western blot analysis increased only slightly in these cells, supporting the observation that the steady state levels of DNA damage is higher in A-T cells than in normals. When treated with PARP inhibitors 3-aminobenzamide or 1,5-dihydroisoquinoline, cellular growth rates reached those observed in normal fibroblast cultures. The improvement of cellular growth and NAD+ levels in A-T cells with PARP inhibition suggests that the cellular metabolic status of A-T cells is compromised and the inhibition of PARP may relieve some of the drain on cellular pyridine nucleotides and ATP. Thus, therapy utilizing PARP inhibitors may provide a benefit for individuals affected with A-T.     

Cleavage of poly(ADP-ribose) polymerase during apoptosis induced by nicotinamide at high concentrations in tobacco suspension cells

Apoptosis induced by high concentrations of nicotinamide in tobacco suspension cells was observed. When cells were treated with 250 mM nicotinamide for 24 h, the hallmarks of apoptosis were detected, including DNA fragments increasing in size by multiples of 180–200 bp, condensation and peripheral distribution of nuclei chromatin and positive reaction to the TUNEL assay. In addition, the degradation of poly (ADP-ribose) polymerase (PARP) was also detected. This indicates that caspase-3-like activity is involved in apoptosis in cultured tobacco cells induced by high-concentration nicotinamide. However, as an inhibitor of PARP, nicotinamide has a contrary effect on apoptosis at low concentrations, which suggests that nicotinamide plays a dual role depending on to its concentration in cells.     

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.