Coelenterazine (marine bioluminescent substrate): a source of inspiration for the discovery of novel antioxidants.

Coelenterazine and derivatives were initially considered in the scientific community for their (bio)luminescent properties. Now, another interest of such hetero-bicycles has been pointed out by the discovery of remarkable antioxidative properties, and an unique mode of action as a "cascade": the mother-compound (imidazolopyrazinone) is transformed by ROS into a daughter-compound (2-amino-pyrazine) also endowed with antioxidative properties. This review illustrates the therapeutic potential of synthetic imidazolopyrazinones (coelenterazine analogues): chemical reactivity assays with singulet oxygen, radical anion superoxide, peroxynitrite, and radicals formed during lipid and LDL peroxidation, cellular tests of protection against oxidative stress using keratinocyte, hepatocyte, neuronal and erythrocyte cells, and finally in vivo evaluation in a hamster model of ischemia-reperfusion, are fully described.

Protection against nitrofurantoin-induced oxidative stress by coelenterazine analogues and their oxidation products in rat hepatocytes.

Coelenterazine (3,7-dihydro-2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-8-benzylimidazolo[1,2-a]pyrazin-3- one) is a substrate for the bioluminescence reaction in many marine animals. Recent work showed that CLZn, its synthetic analogue CLZm, and their common oxidation product coelenteramine (CLM) have strong antioxidative properties in acellular lipid peroxidation systems as well as in rat hepatocytes subjected to tert-butyl hydroperoxide (t-BHP). Here, we analyzed the ability of CLZm and several imidazolopyrazinone (IMPZs) analogues to protect primary cultures of rat hepatocytes against a nitrofurantoin (NF)-induced oxidative stress. Comparison of protection capabilities with reference antioxidants yielded the following ranking: CLZm >>> BHT >Trolox C((R)) > probucol > alpha-tocopherol. The comparison of CLZm with analogues lacking the phenol group in R(1) revealed no differences although the presence of this phenol conferred superior protection against t-BHP. CLM, as well as its methoxylated analogue mCLM which lacks chain-breaking properties, were equally potent in preventing cellular damage caused by NF. mCLM and alpha-naphthoflavone, an inhibitor of cytochrome P450 (CYP450) IAI, similarly protected cells against NF-induced mortality and also equally inhibited EROD activity in methylcholanthrene-induced hepatocytes. The inhibition of EROD by CLZm and CLM was less pronounced. We suggest that the extent of protection conferred by IMPZs against NF-toxicity reflects both the occurrence of antioxidative properties detoxifying ROS produced within cells and inhibitory actions on CYP450 isoforms involved in the bioreduction of NF.

Antioxidative properties of natural coelenterazine and synthetic methyl coelenterazine in rat hepatocytes subjected to tert-butyl hydroperoxide-induced oxidative stress.

Coelenterazine (CLZn; 3, 7-dihydro-2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-8-benzylimidazolo++ +[1 ,2-a]pyrazin-3-one), the substrate for bioluminescence reactions in many marine animals, is endowed with high antioxidant properties. This work investigated the antioxidative properties of CLZn in primary cultures of rat hepatocytes subjected to the oxidant tert-butyl hydroperoxide (t-BHP). Micromolar concentrations of CLZn increased survival and decreased lipid peroxidation in rat hepatocytes subjected for 6 hr to 2.5 x 10(-4) M t-BHP. However, the extent of protection was limited by a strong toxicity of CLZn (IC(50) = 6.9 x 10(-5) M). The presence of t-BHP increased the cellular toxicity of CLZn. Methyl coelenterazine (CLZm, 3, 7-dihydro-2-methyl-6-(p-hydroxyphenyl)-8 benzylimidazolo[1, 2-a]pyrazin-3-one), a synthetic analogue of CLZn, demonstrated excellent antioxidant properties, even at very low (3 x 10(-6) M) concentrations and was not toxic throughout most of its effective concentration range. CLZm proved far more effective than reference antioxidants such as Trolox C(R), alpha-tocopherol, BHT, and probucol. The assay of thiobarbituric reactive substances (TBARS) associated with cells and in the culture medium indicated that 10(-5) M CLZm provided a total protection against t-BHP-induced lipid peroxidation. This coelenterazine analogue could be used as a model compound for investigating the action mechanism of imidazolopyrazinones in mammalian hepatocytes.

Inhibition of the phosphoinositide 3-kinase pathway induces a senescence-like arrest mediated by p27Kip1.

A senescence-like growth arrest is induced in mouse primary embryo fibroblasts by inhibitors of phosphoinositide 3-kinase (PI3K). We observed that senescence-like growth arrest is correlated with an increase in p27(Kip1) but that down-regulation of other cyclin-dependent kinase (CDK) inhibitors, including p15(INK4b), p16(INK4a), p19( INK4d), and p21(Cip1) as well as other negative cell cycle regulators such as p53 and p19(ARF), implies that this senescence-related growth arrest is independent of the activity of p53, p19(ARF), p16(INK4a), and p21(Cip1), which are associated with replicative senescence. The p27(Kip1) binds to the cyclin/CDK2 complexes and causes a decrease in CDK2 kinase activity. We demonstrated that ectopic expression of p27(Kip1) can induce permanent cell cycle arrest and a senescence-like phenotype in wild-type mouse embryo fibroblasts. We also obtained results suggesting that the kinase inhibitors LY294002 and Wortmannin arrest cell growth and induce a senescence-like phenotype, at least partially, through inhibition of PI3K and protein kinase B/Akt, activation of the forkhead protein AFX, and up-regulation of p27(Kip1)expression. In summary, these observations taken together suggest that p27(Kip1) is an important mediator of the permanent cell cycle arrest induced by PI3K inhibitors. Our data suggest that repression of CDK2 activity by p27(Kip1) is required for the PI3K-induced senescence, yet mouse embryo fibroblasts derived from p27(Kip1-/-) mice entered cell cycle arrest after treatment with LY294002. We show that this is due to a compensatory mechanism by which p130 functionally substitutes for the loss of p27(Kip1). This is the first description that p130 may have a role in inhibiting CDK activity during senescence.