Poly(ADP‐ribose)polymerase 1 inhibition protects against age‐dependent endothelial dysfunction

Age‐related endothelial dysfunction is closely associated with the local production of reactive oxygen species (ROS) within and in the vicinity of the vascular endothelium. Oxidant‐induced DNA damage can activate the nuclear enzyme poly(ADP‐ribose) polymerase 1 (PARP‐1), leading to endothelial dysfunction in various pathophysiological conditions. The present study aimed to investigate the role of PARP‐1 in age‐dependent changes in endothelial cell function and its underlying mechanism. Wild‐type (WT) and PARP‐1^?/? mice were divided into young (2 months) and old (12 months) groups. Isolated aortic rings were suspended to record isometric tension to assess endothelial function. Nitric oxide (NO) production and content in plasma were detected by spectrophotometry. Superoxide ( production was detected by dihydroethidium. Expression of PARP‐1, endothelial nitric oxide synthase (eNOS), induced nitric oxide synthase (iNOS), and arginase‐2 (Arg2) was assessed by western blot analysis. Endothelium‐dependent relaxation in response to acetylcholine was lost in old WT, but not PARP‐1^?/?, mice. Endothelium‐independent vasodilation was not impaired in aging mice. Production of was greater in aging WT mice than young or aging PARP‐1^?/? mice. eNOS expression was not affected by aging in WT or PARP‐1^?/? mice, but p‐eNOS expression decreased and iNOS and Arg2 levels were upregulated only in aging WT mice. In conclusion, PARP‐1 inhibition may protect against age‐dependent endothelial dysfunction, potentially by regulating NO bioavailability via iNOS. Inhibition of PARP‐1 may help in vascular aging prevention.     

Effects of a newly developed tricyclic PARP‐1 inhibitor,on ischemic stroke

Poly(ADP‐ribose) polymerase (PARP)‐1 plays an important role in the pathogenic mechanism of ischemic stroke. A number of studies have been undertaken to develop PARP‐1 inhibitors for clinical use. We report on the newly developed PARP‐1 inhibitors, among which 12a showed good activity (IC₅₀=7.8 nM in an enzyme‐based assay and=0.73 µM in a cell‐based assay) and pharmacokinetic profiles. Treatment of the middle cerebral artery (MCA) occluded rats with 3 mg/kg 12a reduced infarct volume suggesting that, may be a good candidate for the treatment of ischemic stroke. Drug Dev Res 71: 253–260, 2010. © 2010 Wiley‐Liss, Inc.     

Contrasting sirtuin and poly(ADP‐ribose)polymerase activities of selected 2,4,6‐trisubstituted benzimidazoles

Both sirtuin and poly(ADP‐ribose)polymerase (PARP) family of enzymes utilize NAD⁺ as co‐substrate. Inhibitors of sirtuins and PARPs are important tools in drug discovery as they are reported to be linked to multiple diseases such as cancer. New potent sirtuin inhibitors (2,4,6‐trisubstituted benzimidazole) were discovered from reported PARP inhibitor scaffold. Interestingly, the synthesized compounds have contrasting sirtuin and PARP‐1 inhibitory activities. We showed that modification on benzimidazoles may alter their selectivity toward sirtuin or PARP‐1 enzymes. This offers an opportunity for further discovery and development of new promising sirtuin inhibitors. Molecular docking studies were carried out to aid the rationalization of these observations. Preliminary antiproliferative studies of selected compounds against nasopharyngeal cancer cells also showed relatively promising results.     

Antagonistic effect of N‐ethylmaleimide on arsenic‐mediated oxidative stress‐induced poly(ADP‐ribosyl)ation and cytotoxicity

Long‐term exposure to arsenic has been known to induce neoplastic initiation and progression in several organs; however, the role of arsenic (As₂O₃) in oxidative stress‐mediated DNA damage remains elusive. One of the immediate cellular responses to DNA damage is poly(ADP‐ribosyl)ation (PARylation), which mediates DNA repair and enhances cell survival. In this study, we found that oxidative stress (H₂O₂)‐induced PARylation was suppressed by As₂O₃ exposure in different human cancer cells. Moreover, As₂O₃ treatment promoted H₂O₂‐induced DNA damage and apoptosis, leading to increased cell death. We found that N‐ethylmaleimide (NEM), an organic compound derived from maleic acid, could reverse As₂O₃‐mediated effects, thus enhancing PARylation with attenuated cell death and increased cell survival. Pharmacologic inhibition of glutathione with l ‐buthionine‐sulfoximine blocked the antagonistic effect of NEM on As₂O₃, thereby continuing As₂O₃‐mediated suppression of PARylation and causing DNA damage. Our findings identify NEM as a potential antidote against As₂O₃‐mediated DNA damage in a glutathione‐dependent manner. Copyright © 2016 John Wiley & Sons, Ltd.     

The neuroprotective effects of the decahydroisoquinoline, LY 215490; a novel AMPA antagonist in focal ischaemia

LY 215490 (3 RS,4aRS,6RS,8aRS)-6-[2-(1(2)H- tetrazole- 5-yl)ethyl]decahydroisoquinoline -3-carboxylic acid), a novel, selective, competitive and systemically active AMPA receptor antagonist was tested as a neuroprotective agent against focal ischaemia in a model of permanent MCA occlusion in the rat. LY 215490 was administered at a dose of 10, 30 or 100 mg/kg 30 min prior to and post-MCA occlusion. The animals were allowed to survive for 24 hr, following which time the brains were processed for volumetric analysis of the infarct size. The low dose of LY 215490 was not effective against the infarct volume in the hemisphere, cortex or caudate. The 2 × 30 mg/kg dose of LY 215490 resulted in 25 and 31% protection against the volume of hemispheric and cortical ischaemic damage, respectively. The highest dose of LY 215490 resulted in a reduced neuroprotective effect with 23 and 27% protection against the volume of hemispheric and cortical ischaemic damage, respectively. The slightly reduced neuroprotective effect of the highest dosing regimen may be due to the respiratory problems seen with this dose. Neither of the two neuroprotective doses of LY 215490 produced any reduction in the volume of caudate damage which represents the core of the infarct.     

Poly(ADP‐ribose) polymerase 1 deficiency increases nitric oxide production and attenuates aortic atherogenesis through downregulation of arginase II

Poly (ADP ‐ribose) polymerase (PARP ) plays an important role in endothelial dysfunction, leading to atherogenesis and vascular‐related diseases. However, whether PARP regulates nitric oxide (NO ), a key regulator of endothelial function, is unclear so far. We investigated whether inhibition of PARP ‐1, the most abundant PARP isoform, prevents atherogenesis by regulating NO production and tried to elucidate the possible mechanisms involved in this phenomenon. In apolipoprotein E‐deficient (apoE^?/?) mice fed a high‐cholesterol diet for 12 weeks, PARP ‐1 inhibition via treatment with 3,4‐dihydro‐54‐(1‐piperindinyl) butoxy‐1(2H)‐isoquinoline (DPQ ) or PARP ‐1 gene knockout reduced aortic atherosclerotic plaque areas (49% and 46%, respectively). Both the groups showed restored NO production in mouse aortas with reduced arginase II (Arg II ) expression compared to that in the controls. In mouse peritoneal macrophages and aortic endothelial cells (MAEC s), PARP ‐1 knockout resulted in lowered Arg II expression. Moreover, phosphorylation of endothelial NO synthase (eNOS ) was preserved in the aortas and MAEC s when PARP ‐1 was inhibited. Reduced NO production in vitro due to PARP ‐1 deficiency could be restored by treating the MAEC s with oxidized low‐density lipoprotein treatment, but this effect could not be achieved with peritoneal macrophages, which was likely due to a reduction in the expression of induced NOS expression. Our findings indicate that PARP ‐1 inhibition may attenuate atherogenesis by restoring NO production in endothelial cells and thus by reducing Arg II expression and consequently arginase the activity.     

New inhibitors of poly(ADP-ribose) polymerase (PARP)

Poly(ADP-ribose) polymerase-1 (PARP-1), the most prominent member of the PARP family, is a DNA-binding protein that is activated by nicks in DNA occurring during inflammation, ischaemia, neurodegeneration or cancer therapy. Activated PARP-1 consumes NAD⁺ that is cleaved into nicotinamide and ADP-ribose and polymerises the latter onto nuclear acceptor proteins. This highly energy consuming process is pivotal for the maintenance of genomic stability although over-activation can culminate in cell dysfunction and necrosis. Therefore, PARP-1 is regarded as a promising target for the development of drugs useful in various forms of inflammation, ischaemia–reperfusion injury and as an adjunct in cancer therapy. This review summarises the structural classes of known PARP-1 inhibitors, with a focus on new inhibitors published for this target, between 2002 and July 2004. The chemistry and biological data disclosed in these patent applications are discussed in light of new structural knowledge of the catalytic domain of the PARP family and recent work with potent inhibitors demonstrating the effects of PARP inhibition in various animal disease models.     

Synthesis of a labeled inhibitor of HIV‐1 attachment: 1‐(4‐benzoylpiperazin‐1‐yl)‐2‐(4,7‐dimethoxy‐1H‐pyrrolo[2,3‐c]pyridinyl‐3‐yl‐[U‐14C]ethane‐1,2‐dione,BMS‐488043‐14C

BMS‐488043 is a potent inhibitor of the interaction between HIV‐1 gp120 and the host cell receptor CD4. We prepared the carbon‐14‐labeled version to support preclinical studies of the compound. It was prepared from ethyl [U‐¹⁴C]oxalyl chloride by a sequence using Friedel–Crafts acylation reaction. The overall radiochemical yield was 82% with a purity of >99.9%. Copyright © 2010 John Wiley & Sons, Ltd.     

Serendipitous Discovery of a Prodrug of a PARP‐1 Inhibitor

During SAR development of previously reported pyrrolocarbazole 1 , a potent PARP‐1 inhibitor, compound 14 , was discovered serendipitously to be a prodrug of compound 1 .     

Neuroprotective effects of a novel translocator protein (18 kDa) ligand,ZBD‐2, against focal cerebral ischemia and NMDA‐induced neurotoxicity

Ligands of the translocator protein (18 kDa) (TSPO) have demonstrated rapid anxiolytic efficacy in stress responses and stress‐related disorders. This protein is involved in the synthesis of endogenous neurosteroids including pregnenolone, dehydroepiandrosterone, and progesterone. These neurosteroids promote γ‐aminobutyric acid‐mediated neurotransmission in the central neural system (CNS). A TSPO ligand, N‐benzyl‐N‐ethyl‐2‐(7,8‐dihydro‐7‐benzyl‐8‐oxo‐2‐phenyl‐9H‐purin‐9‐yl) acetamide (ZBD‐2) was recently synthesized. The purpose of the present study was to investigate the neuroprotective effects of ZBD‐2 and. In cultured cortical neurons, treatment with ZBD‐2 attenuated excitotoxicity induced by N‐methyl‐d ‐aspartate (NMDA) exposure. It significantly decreased the number of apoptotic cells by downregulating GluN2B‐containing NMDA receptors (NMDARs), the ratio of Bax/Bcl‐2, and levels of pro‐caspase‐3. Systemic treatment of ZBD‐2 provided significant neuroprotection in mice subjected to middle cerebral artery occlusion. These findings provide direct evidence that neuroprotection by ZBD‐2 is partially mediated by inhibiting GluN2B‐containing NMDA receptor‐mediated excitotoxicity.     

Long-lasting neuroprotection and neurological improvement in stroke models with new, potent and brain permeable inhibitors of poly(ADP-ribose) polymerase

BACKGROUND AND PURPOSES

Thienyl-isoquinolone (TIQ-A) is a relatively potent PARP inhibitor able to reduce post-ischaemic neuronal death in vitro. Here we have studied, in different stroke models in vivo, the neuroprotective properties of DAMTIQ and HYDAMTIQ, two TIQ-A derivatives able to reach the brain and to inhibit PARP-1 and PARP-2.

EXPERIMENTAL APPROACH

Studies were carried out in (i) transient (2 h) middle cerebral artery occlusion (tMCAO), (ii) permanent MCAO (pMCAO) and (iii) electrocoagulation of the distal portion of MCA in conjunction with transient (90 min) bilateral carotid occlusion (focal cortical ischaemia).

KEY RESULTS

In male rats with tMCAO, HYDAMTIQ (0.1–10 mg·kg⁻¹) injected i.p. three times, starting 4 h after MCAO, reduced infarct volumes by up to 70%, reduced the loss of body weight by up to 60% and attenuated the neurological impairment by up to 40%. In age-matched female rats, HYDAMTIQ also reduced brain damage. Protection, however, was less pronounced than in the male rats. In animals with pMCAO, HYDAMTIQ administered 30 min after MCAO reduced infarct volumes by approximately 40%. In animals with focal cortical ischaemia, HYDAMTIQ treatment decreased post-ischaemic accumulation of PAR (the product of PARP activity) and the presence of OX42-positive inflammatory cells in the ischaemic cortex. It also reduced sensorimotor deficits for up to 90 days after MCAO.

CONCLUSION AND IMPLICATIONS

Our results show that HYDAMTIQ is a potent PARP inhibitor that conferred robust neuroprotection and long-lasting improvement of post-stroke neurological deficits.     

Protective effect of donepezil in primary-cultured rat cortical neurons exposed to N-methyl-d-aspartate (NMDA) toxicity

Donepezil has a neuroprotective effect against oxygen-glucose deprivation injury and glutamate toxicity in cultured cortical neurons. In this study, we further characterized the neuroprotective properties of donepezil in rat cortical cell cultures using glutamate receptor-specific agonists (N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and kainate). Pretreatment with donepezil (1 microM) for 12 h significantly decreased the lactate dehydrogenase (LDH) release in response to NMDA (100 microM) by 43.8%, and reduced the LDH release in response to kainate (100 microM) and AMPA (100 microM) by 11.9% and 7.5% (without statistical significance), respectively. Donepezil appeared to inhibit LDH release in a concentration-dependent manner at 0.1-10 microM. Cortical neurons exposed to NMDA retained a normal morphological appearance in the presence of 10 microM donepezil. In binding assay for glutamate receptors, donepezil at 100 microM only slightly inhibited binding to the glycine and polyamine sites on NMDA receptor complex. On the other hand, 12 h pretreatment with donepezil at 10 and 100 microM significantly decreased the NMDA-induced increase of intracellular calcium concentration ([Ca2+]i). In conclusion, our results show that donepezil has protective activity against NMDA toxicity in cortical neurons, and this neuroprotection seems to be partially mediated by inhibition of the increase of [Ca2+]i.     

A co‐drug conjugate of naringenin and lipoic acid mediates neuroprotection in a rat model of oxidative stress

Using our in vitro and in vivo models of oxidative stress, the current study was designed to determine the neuroprotective potential of naringenin, alone or in combination with lipoic acid. In our mixed neuronal culture exposed to hypoxia and subsequent reoxygenation, naringenin was shown to provide significant neuroprotection against cell death at a concentration of 2.5 μmol/L. Lipoic acid (LA) did not produce neuroprotection at any concentration tested (0.25‐100 μmol/L). In contrast, when naringenin was covalently combined with LA, producing a novel compound named “VANL‐100”, significant neuroprotection was observed at a concentration as low as 2×10^?2 μmol/L (100‐fold more potent). An ELISA for antioxidant capacity demonstrated that naringenin and VANL‐100 likely resulted in neuroprotection by increasing the free radical scavenging capacity of the neuronal cells. Pretreatment of rats with the above compounds prior to middle cerebral artery occlusion (MCAO) followed by reperfusion, showed similar results. Naringenin significantly reduced infarct volume at a dose of 10 mg/kg while VANL‐100 produced significant neuroprotection at a dose as low as 1×10^?4 mg/kg (10 000‐fold more potent). This VANL‐100‐induced neuroprotection persisted even when administered 1 and 3 hours into the reperfusion time course. Taken together, these results suggest that our novel compound, VANL‐100 is neuroprotective, likely via a mechanism that involves increasing the antioxidant capacity of neuronal cells. Our results also show that VANL‐100 is 100‐10 000‐fold more potent than the parent compounds, which adds to the growing evidence in support of combination therapy targeting oxidative stress in neurodegenerative diseases.     

Selective inhibitory effects of HYIpro‐3‐1 on CYP1A2 in human liver microsomes

CYP1A2 is one of the main Cytochrome P450 enzymes in the human liver associated with the metabolism of several xenobiotics. CYP1A2 is especially involved in the metabolic activation of different procarcinogens. Therefore, the development of cancer may be inhibited by inhibiting CYP1A2 activity. Here, the inhibitory effect of HYIpro‐3‐1 and its derivatives on CYP1A2 activity in human liver microsomes (HLM) was studied through LC‐MS/MS using a cocktail assay. Among the four compounds, HYIpro‐3‐1 showed the most selective and strongest inhibitory effect on CYP1A2 at IC₅₀ values of 0.1 µM in HLMs and inhibition was confirmed using purified human CYP1A2. It was determined that inhibition is reversible because the inhibitory effect of HYIpro‐3‐1 is not dependent on preincubation time. HYIpro‐3‐1 showed a typical pattern of competitive inhibition for CYP1A2‐catalyzed phenacetin O‐deethylation, based on the Lineweaver‐Burk plot, with a Ki value of 0.05 μM in HLMs; the secondary plot also showed a linear pattern. In our study, HYIpro‐3‐1 was proposed as a novel inhibitor with the capacity to selectively inhibit CYP1A activity in HLMs.     

Synthesis of 18F‐labeled cyclooxygenase‐2 (COX‐2) inhibitor as a potential PET imaging agent

A new PET tracer for COX‐2 imaging, the 6‐ethoxy‐3‐(4‐methanesulfonylphenyl)‐4‐(4‐[¹⁸F]fluorophenyl)pyran‐2‐one ([¹⁸F]EFMP), was synthesized. For F‐18 radiolabeling, a trimethylammonium precursor and a brominated precursor were synthesized from 1,1,2,3‐tetrachlorocycloprop‐2‐ene in 6 steps. The radiolabeling was achieved through nucleophilic substitution using no‐carrier‐added (n.c.a.) fluorine‐18. Solid‐phase extraction and semi‐preparative‐HPLC purification produced [¹⁸F]EFMP in 14.6±3.3% (n =4) decay corrected radiochemical yield with a specific activity of 487±85.1 (n =4) Ci/mmol and greater than 98% radiochemical purity. Copyright © 2006 John Wiley & Sons, Ltd.