A Novel and Potent Poly(ADP-Ribose) Polymerase-1 Inhibitor, FR247304 (5-Chloro-2-[3-(4-phenyl-3,6-dihydro-1(2H)-pyridinyl)propyl]-4(3H)-quinazolinone), Attenuates Neuronal Damage in in Vitro and in Vivo Models of Cerebral Ischemia

Abstract

The activation of poly(ADP-ribose) polymerase-1 (PARP-1) after exposure to nitric oxide or oxygen-free radicals can lead to cell injury via severe, irreversible depletion of NAD. Genetic deletion or pharmacological inhibition of PARP-1 attenuates brain injury after focal ischemia and neurotoxicity in several neurodegenerative models in animals. FR247304 (5-chloro-2-[3-(4-phenyl-3,6-dihydro-1(2H)-pyridinyl)propyl]-4(3H)-quinazolinone) is a novel PARP-1 inhibitor that has recently been identified through structure-based drug design. In an enzyme kinetic analysis, FR247304 exhibits potent and competitive inhibition of PARP-1 activity, with a K i value of 35 nM. Here, we show that prevention of PARP activation by FR247304 treatment protects against both reactive oxygen species-induced PC12 cell injury in vitro and ischemic brain injury in vivo. In cell death model, treatment with FR247304 (10 Ϫ8 -10 Ϫ5 M) significantly reduced NAD depletion by PARP-1 inhibition and attenuated cell death after hydrogen peroxide (100 M) exposure. After 90 min of middle cerebral artery occlusion in rats, poly(ADP-ribosy)lation and NAD depletion were markedly increased in the cortex and striatum from 1 h after reperfusion. The increased poly(ADPribose) immunoreactivity and NAD depletion were attenuated by FR247304 (32 mg/kg i.p.) treatment, and FR247304 significantly decreased ischemic brain damage measured at 24 h after reperfusion. Whereas other PARP inhibitors such as 3-aminobenzamide and PJ34 [N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylactamide] showed similar neuroprotective actions, they were less potent in in vitro assays and less efficacious in an in vivo model compared with FR247304. These results indicate that the novel PARP-1 inhibitor FR247304 exerts its neuroprotective efficacy in in vitro and in vivo experimental models of cerebral ischemia via potent PARP-1 inhibition and also suggest that FR247304 or its derivatives could be attractive therapeutic candidates for stroke and neurodegenerative disease.Activation of nuclear enzyme poly(ADP-ribose) polymerase (PARP) promotes cell death through processes involving energy depletion. Reactive oxygen species (ROS)-mediated damage of DNA can activate PARP (Szabo et al., 1996;Eliasson et al., 1997) and consumes NAD and consequently ATP, culminating in cell dysfunction or necrosis (Ha and Snyder, 1999). In addition, PARP plays a central role in the caspase-independent apoptosis pathway mediated by apoptosis-inducing factor. Translocation of apoptosis-inducing factor from the mitochondria to the nucleus is dependent on PARP activation in neurons treated with various DNA-damaging stimuli, including hydrogen peroxide (Yu et al., 2002). This cellular suicide mechanism of both necrosis and apoptosis by PARP activation has been implicated in the pathogenesis of ischemic brain injury and neurodegenerative disorders, and PARP inhibitors have been shown to be effective in animal models of stroke, traumatic brain injury and Parkinson's disease (Cosi et al., 1996;Endres et al...

Protein Kinase C Epsilon Regulates Mitochondrial Pools of Nampt and NAD Following Resveratrol and Ischemic Preconditioning in the Rat Cortex

“…During ischemia, overactivation of PARPs mediates NAD þ depletion and apoptotic signaling. 3,4 Protein kinase C epsilon-mediated increases in mitochondrial NAD þ availability may help to maintain pools of NAD þ during activity of mitochondrial-localized PARPs (Figure 7), thereby maintaining mitochondrial DNA integrity and preventing cell death. 18,39 The protective role of Nampt against neurodegenerative damage has recently become the focus of intense investigation.…”

“…16 Reductions in NAD during ischemic events cause DNA damage, energy depletion and lead to neurodegeneration. [4][5][6]37 Nicotinamide phosphoribosyltransferase overexpression has been shown to prevent mitochondrial dysfunction and neuronal death after ischemia through its ability to produce NAD. 6,15 Specifically enhancing mitochondriallocalized NAD after oxidative stress was shown to preserve mitochondrial membrane potential, enhanced respiration, and prevented the release of apoptosis-inducing factor in neurons.…”

“…1 In the brain, reductions in NAD after oxidative stress or ischemic events cause mitochondrial depolarization, decreased respiration, apoptotic signaling, and neuronal death. [2][3][4] Conversely, increases in cellular NAD have been shown to prevent mitochondrial dysfunction and neurodegeneration after cerebral ischemia. 5,6 Ischemic preconditioning (IPC), a paradigm where a brief ischemic insult protects the brain against a subsequent lethal ischemic injury, or treatment with the polyphenol resveratrol, has been shown to protect mitochondria and enhance levels of NAD in the brain.…”

Pathways for Ischemic Cytoprotection: Role of Sirtuins in Caloric Restriction, Resveratrol, and Ischemic Preconditioning

“…Increasing NAD + levels before or after OGD in neurons protects against DNA strand breaks and apurinic/apyrimidinic abasic sites, while also enhancing the activity of enzymes that repair damaged DNA . After cerebral ischemia, there is a significant reduction in NAD + in the brain (Iwashita et al, 2004), which may have a major role in cell death caused by genotoxic stress (Yang et al, 2007a). Little is known about the mechanism by which NAD + is synthesized in the mitochondria.…”

“…Replenishing NAD + before or after OGD in primary neuronal cultures was shown to markedly reduce ischemic injury . Similarly, inhibition of poly(ADP-ribose) polymerase 1, an enzyme activated by DNA damage that consumes cellular NAD + stores, inhibited NAD + depletion and prevented ischemic injury after middle cerebral artery occlusion (Iwashita et al, 2004). Like poly(ADP-ribose) polymerase 1, SIRT1 requires NAD + as a cofactor for enzymatic activity.…”