Neurounina-1, a novel compound that increases Na+/Ca 2+ exchanger activity, effectively protects against stroke damage

Pasquale Molinaro, Maria Cantile, Ornella Cuomo, Agnese Secondo, Anna Pannaccione, Paolo Ambrosino, Giuseppe Pignataro, Ferdinando Fiorino, Beatrice Severino, Elena Gatta, Maria José Sisalli, Marco Milanese, Antonella Scorziello, Giambattista Bonanno, Mauro Robello, Vincenzo Santagada, Giuseppe Caliendo, Gianfranco Di Renzo, Lucio Annunziato

Research output: Contribution to journalArticle

Abstract

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 EC 50 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 a-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.

Original languageEnglish
Pages (from-to)142-156
Number of pages15
JournalMolecular Pharmacology
Volume83
Issue number1
DOIs
Publication statusPublished - Jan 2013

ASJC Scopus subject areas

  • Pharmacology
  • Molecular Medicine

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    Molinaro, P., Cantile, M., Cuomo, O., Secondo, A., Pannaccione, A., Ambrosino, P., Pignataro, G., Fiorino, F., Severino, B., Gatta, E., Sisalli, M. J., Milanese, M., Scorziello, A., Bonanno, G., Robello, M., Santagada, V., Caliendo, G., Di Renzo, G., & Annunziato, L. (2013). Neurounina-1, a novel compound that increases Na+/Ca 2+ exchanger activity, effectively protects against stroke damage. Molecular Pharmacology, 83(1), 142-156. https://doi.org/10.1124/mol.112.080986