New inhibitors of poly(ADP-ribose) polymerase and their potential therapeutic targets

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding protein that is activated by nicks in the DNA molecule. It regulates the activity of various enzymes, including itself, that are involved in the control of DNA metabolism. Evidence obtained with both benzamide and isoquinolinone PARP inhibitors and the PARP-1(^-/-) phenotype, clearly indicate that PARP plays an important role in NO/ROS-induced cell damage during inflammation, ischaemia and neurodegeneration. PARP is involved in the maintenance of genomic stability and PARP inhibition may also potentiate the cytotoxic action of agents used in cancer therapy. Benzamides, although not very potent (IC₅₀ ~ 20 – 50 μM) PARP inhibitors, have been widely used to probe PARP functions, because of their lack of toxicity both in vitro and in vivo, even at high doses. In the early 1990s, a new class of very potent PARP inhibitors (i.e., at least 100-fold more potent thatn benzamide), the dihydroisoquinolinones, benzamide derivatives with the carbamoyl group constrained into the antiorientation, was discovered. At the same time, a large structure–activity surevey identified over 13 chemical classes of PARP inhibitors, the most potent calss sharing a common structural feature, the presence of a carbonyl group built into a polyaromatic heterocyclic skeleton or a carbamoyl group attached to an aromatic ring. Recently, a better knowledge of the PARP catalytic domain and the use of its crystal structure have led to the design and synthesis of the tricyclic lactam indoles, active at low nanomolar concentrations, and with favourable physical properties and in vivo characteristics. In the last few years the interest in PARP as a therapeutic target has been rapidly growing. This article reviews the patents filed for new PARP inhibitors over the last three years, up to February 2002, and their development status.     

Role of poly(ADP-ribose) polymerase 1 (PARP-1) in cardiovascular diseases: the therapeutic potential of PARP inhibitors

Accumulating evidence suggests that the reactive oxygen and nitrogen species are generated in cardiomyocytes and endothelial cells during myocardial ischemia/reperfusion injury, various forms of heart failure or cardiomyopathies, circulatory shock, cardiovascular aging, diabetic complications, myocardial hypertrophy, atherosclerosis, and vascular remodeling following injury. These reactive species induce oxidative DNA damage and consequent activation of the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1), the most abundant isoform of the PARP enzyme family. PARP overactivation, on the one hand, depletes its substrate, NAD+, slowing the rate of glycolysis, electron transport, and ATP formation, eventually leading to the functional impairment or death of the endothelial cells and cardiomyocytes. On the other hand, PARP activation modulates important inflammatory pathways, and PARP-1 activity can also be modulated by several endogenous factors such as various kinases, purines, vitamin D, thyroid hormones, polyamines, and estrogens, just to mention a few. Recent studies have demonstrated that pharmacological inhibition of PARP provides significant benefits in animal models of cardiovascular disorders, and novel PARP inhibitors have entered clinical development for various cardiovascular indications. Because PARP inhibitors can enhance the effect of anticancer drugs and decrease angiogenesis, their therapeutic potential is also being explored for cancer treatment. This review discusses the therapeutic effects of PARP inhibitors in myocardial ischemia/reperfusion injury, various forms of heart failure, cardiomyopathies, circulatory shock, cardiovascular aging, diabetic cardiovascular complications, myocardial hypertrophy, atherosclerosis, vascular remodeling following injury, angiogenesis, and also summarizes our knowledge obtained from the use of PARP-1 knockout mice in the various preclinical models of cardiovascular diseases.     

Tricyclic benzimidazoles as potent poly(ADP-ribose) polymerase-1 inhibitors

Novel tricyclic benzimidazole carboxamide poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been synthesized. Several compounds were found to be powerful chemopotentiators of temozolomide and topotecan in both A549 and LoVo cell lines. In vitro inhibition of PARP-1 was confirmed by direct measurement of NAD+ depletion and ADP-ribose polymer formation caused by chemically induced DNA damage.     

NGF promotes long-term memory formation by activating poly(ADP-ribose)polymerase-1

Nerve growth factor (NGF) is a critical secreted protein that plays an important role in development, survival, and function of the mammalian nervous system. Previously reports suggest that endogenous NGF is essential for the hippocampal plasticity/memory and NGF deprivation induces the impairment of hippocampus-related memory and synaptic plasticity. However, whether exogenous supplement of NGF could promote the hippocampus-dependent synaptic plasticity/memory and the possible underlying mechanisms are not clear. In this study we found that NGF administration facilitates the hippocampus-dependent long-term memory and synaptic plasticity by increasing the activity of PARP-1, a polymerase mediating the PolyADP-ribosylation and important for the memory formation. Co-application of 3-Aminobenzamide (3-AB), a specific inhibitor of PARP-1, distinctly blocked the boosting effect of NGF on memory and synaptic plasticity, and the activation of downstream PKA-CREB signal pathway. Our data provide the first evidence that NGF supplement facilitates synaptic plasticity and the memory ability through PARP-1-mediated protein polyADP-ribosylation and activation of PKA-CREB pathway.     

A dual role for poly(ADP-ribose) polymerase-1 during caspase-dependent apoptosis

2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, catalyzes the generation of reactive oxygen species (ROS) and caspase-dependent apoptosis in human promyelocytic leukemia (HL-60) cells. We now report that TGHQ induces severe DNA damage, as evidenced by DNA ladder formation and H2AX phosphorylation. The subsequent activation of the DNA nick sensor enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), leads to the rapid depletion of ATP and NAD and the concomitant formation of poly(ADP-ribosylated) proteins (PARs). PJ-34 (a PARP-1 inhibitor) completely prevented the formation of PARs, partially attenuated TGHQ-mediated ATP depletion, but had little effect on NAD depletion. Intriguingly, although z-vad-fmk (a pan-caspase inhibitor) attenuated TGHQ-induced apoptosis, cotreatment with PJ-34 led to a further decrease in apoptosis, suggesting that PARP-1 participates in caspase-dependent apoptosis. Indeed, PARP-1 inhibition reduced TGHQ-induced caspase-3, -7, and -9 activation, at least partially by attenuating cytochrome c translocation from mitochondria to the cytoplasm. In contrast, PJ-34 potentiated TGHQ-induced caspase-8 activation, suggesting that PARP-1 plays a dual role in regulating TGHQ-induced apoptosis via opposing effects on the intrinsic (mitochondrial) and extrinsic (death-receptor) pathways. PARP-1 knockdown in HL-60 cells confirmed that PARP-1 participates in effector caspase activation. Finally, PJ-34 also inhibited TGHQ-induced apoptosis-inducing factor (AIF) nuclear translocation, but neither c-jun NH(2)-terminal kinase nor p38 MAPK (p38 mitogen-activated protein kinase) activation was required for AIF translocation. In summary, TGHQ-induced apoptosis of HL-60 cells is accompanied by PARP-1, caspase activation, and AIF nuclear translocation. TGHQ-induced apoptosis appears to primarily occur via engagement of the mitochondrial-mediated pathway in a process amenable to PARP inhibition. Residual cell death in the presence of PJ-34 is likely mediated via the extrinsic apoptotic pathway.     

Role of the poly(ADP-ribose)polymerase activity in vancomycin-induced renal injury

The aim of the present study was to investigate the role of poly(ADP-ribose)polymerase (PARP) activity in vancomycin (VCM)-induced renal injury and to determine whether 1,5-isoquinelinediol (ISO), a PARP inhibitor agent, could be offered as an alternative therapy in VCM-induced renal impairment. Rats were divided into four groups as follows: (i) control (Group 1); (ii) VCM-treated (Group 2); (iii) VCM plus ISO-treated (Group 3); and (iv) ISO-treated (Group 4). VCM (200 mg/kg, i.p., twice daily) was administered to Groups 2 and 3 for 7 days. ISO (3 mg/kg/day, i.p.) treatment was started 24 h before the first administration of VCM and continued for 8 days. After the 14th VCM injection, the animals were placed in metabolic cages to collect urine samples. All the rats were sacrificed by decapitation, blood samples were taken in tubes and kidneys were excised immediately. Blood urea nitrogen (BUN) and plasma creatinine, and urinary N-acetyl-β-d-glucosaminidase (NAG, a marker of renal tubular injury) were used as markers of VCM-induced renal injury in rats. Light microscopy was used to evaluate semi-quantitative analysis of the kidney sections. Poly(ADP-ribose) (PAR, the product of activated PARP) and PARP-1 expressions in renal tissues were demonstrated by immunohistochemistry and Western blot. VCM administration increased BUN levels from 8.07 ± 0.75 mg/dL to 53.87 ± 10.11 mg/dL. The plasma creatinine levels were 0.8 ± 0.04 mg/dL and 3.38 ± 0.51 mg/dL for the control and VCM-treated groups, respectively. Also, urinary excretion of NAG was increased after VCM injection. Besides, there was a significant dilatation of the renal tubules, eosinophilic casts within some tubules, desquamation and vacuolization of renal tubule epithelium, and interstitial tissue inflammation in VCM-treated rats. In VCM-treated rats, both PAR and PARP-1 expressions were increased in renal tubular cells. ISO treatment attenuated VCM-induced renal injury, as indicated by BUN and plasma creatinine levels, urinary NAG excretion, and renal histology. PARP inhibitor treatment also decreased PAR and PARP-1 protein expressions similar to that of controls. Herewith, the overactivation of the PARP pathway may have a role in VCM-induced renal impairment and pharmacological inhibition of this pathway might be an effective intervention to prevent VCM-induced acute renal injury.     

汉防己甲素对大鼠脊髓损伤氧自由基的影响

目的观察汉防己甲素（Ｔｅｔ）对急性脊髓损伤（ＡＳＣＩ）的保护作用,并从自由基损伤的角度探讨其对ＡＳＣＩ的作用机理。方法７５只 Ｗｉｓｔａｒ大鼠改良Ａｌｉｅｎ’ｓ打击法复制大鼠全瘫模型后随机分为 ３组：（１）生理盐水（ＮＳ）组;（２）汉防己甲素（Ｔｅｔ）组;（３）甲基强的松龙组（ＭＰ）。伤后 ３０分钟 ｉｖ Ｔｅｔ２２．５ｍｇ／ｋｇ体重。于伤后１小时、４小时、８小时取损伤区脊髓组织进行形态学观察和生化指标测定。结果汉防己甲素能明显降低组织钙、ＭＤＡ含量,防止ＳＯＤ含量的下降,使伤区脊髓组织中水含量减少,防止ＬＤＨ、ＣＰＫ等细胞内物质的泄漏,减轻组织病损。结论Ｔｅｔ对ＡＳＣＩ有保护作用,这一作用机制是通过抑制自白基的生成、减轻脂质过氧化的程度并与其钙拮抗起协同治疗作用。     

Development of a miniaturized assay for the high-throughput screening program for poly(ADP-ribose) polymerase-1

Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, and neurodegenerative disease. With these emerging medicinal interests, various screening programs have identified small molecules that inhibit PARP with reasonable potencies. However, the increasing numbers of diverse small molecules generated through combinatorial chemistry necessitate the use of robust assays with good sensitivity and specificity for use as a high-throughput screening (HTS) program. Here, we report the development and the validation of a nonisotopic PARP-1 assay suitable for HTS by converting a biotinylated NAD-based colorimetric assay to a miniaturized 384-well plate format. Comparing with the conventional methods, this miniaturized PARP-1 inhibition assay was equally sensitive with excellent reproducibility and cost-effectiveness. Because nonisotopic PARP-1 inhibition assays are widely used, the methodology described in this article can expand the feasibility of this assay as a high-throughput assay for screening of PARP-1 inhibitors from a random chemical library.     

Caffeine metabolites are inhibitors of the nuclear enzyme poly(ADP-ribose)polymerase-1 at physiological concentrations

The activity of the nuclear enzyme poly(ADP-ribose)polymerase-1 (E.C.2.4.2.30), which is highly activated by DNA strand breaks, is associated with the pathophysiology of both acute as well as chronic inflammatory diseases. PARP-1 overactivation and the subsequent extensive turnover of its substrate NAD+ put a large demand on mitochondrial ATP-production. Furthermore, due to its reported role in NF-kappaB and AP-1 mediated production of pro-inflammatory cytokines, PARP-1 is considered an interesting target in the treatment of these diseases. In this study the PARP-1 inhibiting capacity of caffeine and several metabolites as well as other (methyl)xanthines was tested using an ELISA-assay with purified human PARP-1. Caffeine itself showed only weak PARP-1 inhibiting activity, whereas the caffeine metabolites 1,7-dimethylxanthine, 3-methylxanthine and 1-methylxanthine, as well as theobromine and theophylline showed significant PARP-1 inhibiting activity. Further evaluation of these compounds in H2O2-treated A549 lung epithelial and RF24 vascular endothelial cells revealed that the decrease in NAD+-levels as well as the formation of the poly(ADP-ribose)polymer was significantly prevented by the major caffeine metabolite 1,7-dimethylxanthine. Furthermore, H2O2-induced necrosis could be prevented by a high dose of 1,7-dimethylxanthine. Finally, antioxidant effects of the methylxanthines could be ruled out with ESR and measurement of the TEAC. Concluding, caffeine metabolites are inhibitors of PARP-1 and the major caffeine metabolite 1,7-dimethylxanthine has significant PARP-1 inhibiting activity in cultured epithelial and endothelial cells at physiological concentrations. This inhibition could have important implications for nutritional treatment of acute and chronic inflammatory pathologies, like prevention of ischemia-reperfusion injury or vascular complications in diabetes.     

Drug targets for traumatic brain injury from poly(ADP-ribose)polymerase pathway modulation

The deleterious pathophysiological cascade induced after traumatic brain injury (TBI) is initiated by an excitotoxic process triggered by excessive glutamate release. Activation of the glutamatergic N-methyl-D-aspartate receptor, by increasing calcium influx, activates nitric oxide (NO) synthases leading to a toxic production of NO. Moreover, after TBI, free radicals are highly produced and participate to a deleterious oxidative stress. Evidence has showed that the major toxic effect of NO comes from its combination with superoxide anion leading to peroxynitrite formation, a highly reactive and oxidant compound. Indeed, peroxynitrite mediates nitrosative stress and is a potent inducer of cell death through its reaction with lipids, proteins and DNA. Particularly DNA damage, caused by both oxidative and nitrosative stresses, results in activation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme implicated in DNA repair. In response to excessive DNA damage, massive PARP activation leads to energetic depletion and finally to cell death. Since 10 years, accumulating data have showed that inactivation of PARP, either pharmacologically or using PARP null mice, induces neuroprotection in experimental models of TBI. Thus TBI generating NO, oxidative and nitrosative stresses promotes PARP activation contributing in post-traumatic motor, cognitive and histological sequelae. The mechanisms by which PARP inhibitors provide protection might not entirely be related to the preservation of cellular energy stores, but might also include other PARP-mediated mechanisms that needed to be explored in a TBI context. Ten years of experimental research provided rational basis for the development of PARP inhibitors as treatment for TBI.     

ATP modulates poly(ADP-ribose) polymerase-1-facilitated topoisomerase I-linked DNA religation in the presence of camptothecin

Poly(ADP-ribose) polymerase (PARP)-1 was reported to promote the religation activity of topoisomerase I in the presence of camptothecin by itself through the direct interaction with topoisomerase I or by the formation of poly(ADP-ribosyl)ated topoisomerase I. We have demonstrated previously that ATP inhibited PARP-1/NAD-facilitated religation of topoisomerase I-linked DNA (TLD) in the presence of camptothecin. The mechanism of action was further studied in the present work. ATP as well as other nucleotides, including CTP, UTP, and GTP, had no effect on topoisomerase I cleavage and religation activities in the absence of camptothecin. In the presence of camptothecin or its derivative topotecan, ATP (at up to 2 mM) inhibited PARP-1/NAD-facilitated TLD religation in a dose-dependent manner. This could be due to the suppression of topoisomerase I poly(ADP-ribosyl)ation through the competition with NAD for the binding site(s) on PARP-1. The interaction between ATP and PARP-1 was independent of ATP hydrolysis. Study of different nucleotide analogs revealed that the structure could determine the dose response of nucleotides. In addition, it was noted that higher concentrations of ATP and CTP (at 2.5 mM or higher) promoted DNA religation by a PARP-1-independent mechanism. Our study implies the possible role of ATP and other nucleotides in the regulation of topoisomerase I activity in the presence of camptothecin analogs.     

Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors in cancer chemotherapy

Poly(ADP-ribose) polymerases (PARPs) are defined as a family of cell signaling enzymes present in eukaryotes, which are involved in poly(ADP-ribosylation) of DNA-binding proteins. The best studied of these enzymes (PARP-1) is involved in the cellular response to DNA damage so that in the event of irreparable DNA damage overactivation of PARP-1 leads to necrotic cell death. Inhibitors of PARP-1 activity in combination with DNA-binding antitumor drugs may constitute a suitable strategy in cancer chemotherapy. When DNA is moderately damaged, PARP-1 participates in the DNA repair process and the cell survives. However, in the case of extensive DNA damage PARP-1 overactivation induces a decrease of NAD+ and ATP levels leading to cell dysfunction or even to necrotic cell death. So, due to PARP-1 involvement in cell death, pharmacological inhibition of PARP-1 activity by PARP-1 inhibitors may constitute a suitable target to enhance the activity of antitumor drugs through inhibition of necrosis and activation of apoptosis. PARP-1 inhibitors such as 3-aminobenzamide, 1,5-dihydroxyisoquinolinone and the recently patented tryciclic benzimidazoles have shown potent inhibitory effects of PARP-1 activity in tumor cells. The present review gives an update of the state-of-the-art of inhibition of PARP-1 activity as adjuvant therapy in cancer treatment.     

Oxidative stress initiates DNA damager MNNG-induced poly(ADP-ribose)polymerase-1-dependent parthanatos cell death

The alkylating agent N-methyl-N′-nitro-N′-nitrosoguanidine (MNNG) can cause excess DNA strand breaks that lead to poly(ADP-ribose)polymerase-1 (PARP-1) overactivation and cell death (parthanatos). However, the detail mechanism of MNNG-induced parthanatos was not well-investigated. In this study, we used MNNG-treated mouse embryonic fibroblasts (MEFs) to elucidate the signaling pathways of MNNG-induced parthanatos. We found that MNNG-induced cell death accompanied by rapid PARP-1 activation, c-Jun N-terminal kinase (JNK) activation, biphasic reactive oxygen species (ROS) production and intracellular calcium increase. The early ROS production occurring at 1 min and peaking at 5–15 min after MNNG treatment partially resulted from NADPH oxidase. In contrast, the late phase of ROS production occurring at 30 min and time-dependently increasing up to 6 h after MNNG treatment was generated by mitochondria. The antioxidant, NAC can abrogate all phenomena caused by MNNG. Results indicate that the calcium rise was downstream of early ROS production, and was involved in PARP-1 and JNK activation. Moreover, the PARP inhibitor was able to reduce MNNG-induced late-phase ROS production, calcium elevation, and cell death. Results further indicated the involvement of RIP1 in sustained ROS production and calcium increase. We characterized the interactive roles of ROS, calcium, JNK, and RIP1 in MNNG-induced cell death. We found that in addition to the alkylating property previously demonstrated, ROS production triggered by MNNG results in enhanced DNA damage and PARP-1 activation. Moreover, intracellular calcium elevation and ROS production have mutual amplification effects and thus contribute to PARP-1-mediated parthanatos.     

Role of nitrosative stress and poly(ADP-ribose) polymerase activation in myocardial reperfusion injury

Ischemia and reperfusion injury leads to a complex pathophysiological process, which in turn results in the generation of free radicals. Peroxynitrite, a highly reactive species causes DNA single strand breaks, which activates the nuclear enzyme, poly (ADP-ribose) polymerase (PARP). The activation of PARP leads to an energy consuming inefficient repair cycle with subsequent depletion of NAD(+) and ATP pools and necrotic cell death. The present review overviews the pathophysiological role of the peroxynitrite-PARP pathway in cardiac ischemia/reperfusion injury with special reference to the therapeutic potential of PARP inhibitors in the treatment of this disease.     

Discovery of potent poly(ADP-ribose) polymerase-1 inhibitors from the modification of indeno[1,2-c]isoquinolinone

Novel indeno[1,2-c]isoquinolinone derivatives were synthesized and evaluated as inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). These potent nonmutagenic PARP-1 inhibitors possess an additional five-membered ring between the B and C rings of 6(5H)-phenanthridinone. The most potent PARP-1 inhibitors were obtained from the substitution of the D ring at the C-9 position, in particular sulfonamide and N-acyl analogues (6 and 11). The 9-sulfonamide analogues 11a and 12a exhibited IC(50) values of 1 and 10 nM, respectively.