Akinori Iwashita

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.

Methamphetamine (METH) administration in mice, results in a chronic dopamine (DA) depletion associated with nerve terminal damage, with DA oxidation and generation of reactive oxygen species (ROS) primarily mediating this neurotoxicity. The oxidative stress induced by METH putatively activates nuclear enzyme poly(ADP-ribose) polymerase (PARP), with excessive PARP activation eventually leading to cell death. In this study, we show that prevention of PARP activation by treatment with FR261529 [2-(4-chlorophenyl)-5-quinoxalinecarboxamide], the compound that was recently identified as a novel PARP inhibitor (IC₅₀ for PARP-1 = 33 nM, IC₅₀ for PARP-2 = 7 nM), protects against both ROS-induced cells injury in vitro and METH-induced dopaminergic neuronal damage in an in vivo Parkinson's disease (PD) model. In PC12 cells, exposure of hydrogen peroxide or METH markedly induced PARP activation, and treatment with FR261529 (1 μM) significantly reduced PARP activation and attenuated cell death. In the mouse METH model, METH (15 mg/kg × 2 i.p., 2 h apart) intoxication accelerated DA metabolism and oxidation in the striatum, with subsequent cell damage in nigrostriatal dopaminergic neurons after 4 days. Oral administration of FR261529 (10 or 32 mg/kg) attenuated the damage of dopaminergic neurons via marked reduction of PARP activity and not via changes in dopamine metabolism or body temperature. These findings indicate that the neuroprotective effects of a novel PARP inhibitor, FR261529, were accompanied by inhibition of METH-induced PARP activation, suggesting that METH induces nigrostriatal dopaminergic neurodegeneration involving PARP activation and also orally active and brain-penetrable PARP inhibitor FR261529 could be a novel attractive therapeutic candidate for neurodegenerative disorders such as PD.

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^–8-10^–5 M) significantly reduced NAD depletion by PARP-1 inhibition and attenuated cell death after hydrogen peroxide (100 μM) 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(ADP-ribose) 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.

The massive activation of poly(ADP-ribose) polymerase-1 (PARP-1) by DNA-damaging stimuli, such as exposure to reactive oxygen species (ROS), can lead to cell injury via severe, irreversible depletion of the NAD and ATP pool, and PARP-1 inhibitors have been expected to rescue neurons from degeneration in a number of disease models. We have recently identified 2-{3-[4-(4-chlorophenyl)-1-piperazinyl] propyl}-4(3H)-quinazolinone (FR255595) as a novel and potent PARP-1 inhibitor through structure-based drug design and high-throughput screening. This compound potently inhibited PARP activity with an IC₅₀ value of 11 nM and was orally active and highly brain penetrable. Here, we show that prevention of PARP activation by FR255595 protects against both ROS-induced cells injury in vitro and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal dopaminergic damage in an in vivo Parkinson's disease (PD) model. In cell death models in vitro, exposure of hydrogen peroxide induced cell death with PARP overactivation in PC12 cells and SH-SY5Y cells, and pre- and post-treatment with FR255595 (10^-9-10^-5 M) significantly reduced PARP activation and cell death. In mouse MPTP model, MPTP (20 mg/kg i.p.) intoxication lead to PARP activation and cell damage in the nigrostriatal dopaminergic pathway, which was significantly ameliorated by oral administration of FR255595 (10-32 mg/kg), both in the substantia nigra and in the striatum via marked reduction of PARP activation, even with delayed treatment. These findings clearly indicate that the novel PARP-1 inhibitor FR255595 exerts neuroprotective effect through its potent PARP-1 inhibitory actions in PD model, suggesting that the drug could be an attractive candidate for several neurodegenerative disorders, including PD.

Reactive oxygen species (ROS) play a vital role in brain damage after cerebral ischemia-reperfusion injury, and ROS scavengers have been shown to exert neuroprotective effects against ischemic brain injury. We have recently identified 8-(4-fluorophenyl)-2-((2E)-3-phenyl-2-propenoyl)-1,2,3,4-tetrahydropyrazolo[5,1-c][1,2,4]triazine (FR210575) as a novel, powerful free-radical scavenger. In the present study, the neuroprotective efficacy of FR210575 was evaluated in two neuronal death models in vitro as well as rat focal cerebral ischemia models in vivo. In the first model, primary cortical cultures were exposed to a high oxygen atmosphere (50% O₂) for 48 h to induce cell death with apoptotic features. Treatment with FR210575 (10^–7–10^–5 M) significantly inhibited neuronal death. The second model used a growth-factor withdrawal paradigm. Withdrawal of TIP (transferrin, insulin, putrescine and progesterone)-supplemented medium induced apoptotic cell death after 2 days, but treatment with FR210575 exhibited dramatic protection against neuronal death. In two models of cerebral ischemia [photothrombotic occlusion of middle cerebral artery (MCA) for transient model and by permanent MCA occlusion for permanent model], rats received 3-h intravenous infusion (1–10 mg/kg/3 h) of FR210575, with brain damage determined 24 h later. FR210575 (3.2 mg/kg/3 h) significantly reduced the volume of focal damage in the cortex by 36% in the transient model and also reduced the size of ischemic brain damage in the permanent model. These findings indicate that the powerful radical scavenger FR210575 has potent neuroprotective activity and that FR210575 could be an attractive candidate for the treatment of stroke or other neurodegenerative disorders.