Neurounina-1, a Novel Compound that Increases Na+/Ca2+ Exchanger Activity, Effectively Protects Against Stroke Damage

Previous studies have demonstrated that the knock-down or knock-out of the three Na(+)/Ca(2+) 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. Ca(2+)-radiotracer, Fura-2-microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC(50) in the picomolar-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 10nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced GABA release, enhanced GABA(A)-currents, and inhibited both glutamate release and NMDA receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered i.p. at doses ranging 0.003-30 μg/kg, it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3-5h after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities.