PT - JOURNAL ARTICLE AU - Pasquale Molinaro AU - Maria Cantile AU - Ornella Cuomo AU - Agnese Secondo AU - Anna Pannaccione AU - Paolo Ambrosino AU - Giuseppe Pignataro AU - Ferdinando Fiorino AU - Beatrice Severino AU - Elena Gatta AU - Maria José Sisalli AU - Marco Milanese AU - Antonella Scorziello AU - Giambattista Bonanno AU - Mauro Robello AU - Vincenzo Santagada AU - Giuseppe Caliendo AU - Gianfranco Di Renzo AU - Lucio Annunziato TI - Neurounina-1, a Novel Compound That Increases Na<sup>+</sup>/Ca<sup>2+</sup> Exchanger Activity, Effectively Protects against Stroke Damage AID - 10.1124/mol.112.080986 DP - 2013 Jan 01 TA - Molecular Pharmacology PG - 142--156 VI - 83 IP - 1 4099 - http://molpharm.aspetjournals.org/content/83/1/142.short 4100 - http://molpharm.aspetjournals.org/content/83/1/142.full SO - Mol Pharmacol2013 Jan 01; 83 AB - Previous studies have demonstrated that the knockdown or knockout of the three Na+/Ca2+ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, worsens ischemic brain damage. This suggests that the activation of these antiporters exerts a neuroprotective action against stroke damage. However, drugs able to increase the activity of NCXs are not yet available. We have here succeeded in synthesizing a new compound, named neurounina-1 (7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one), provided with an high lipophilicity index and able to increase NCX activity. Ca2+ radiotracer, Fura-2 microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC50 in the picomolar to low nanomolar range, whereas it did not affect NCX3 activity. Furthermore, by using chimera strategy and site-directed mutagenesis, three specific molecular determinants of NCX1 responsible for neurounina-1 activity were identified in the α-repeats. Interestingly, NCX3 became responsive to neurounina-1 when both α-repeats were replaced with the corresponding regions of NCX1. In vitro studies showed that 10 nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced γ-aminobutyric acid (GABA) release, enhanced GABAA currents, and inhibited both glutamate release and N-methyl-d-aspartate receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered at doses of 0.003 to 30 μg/kg i.p., it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3 to 5 hours after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities.