Pharmacological Characterization of the Newly Synthesized 5-Amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide Hydrochloride (BED) as a Potent NCX3 Inhibitor That Worsens Anoxic Injury in Cortical Neurons, Organotypic Hippocampal Cultures, and Ischemic Brain

The Na⁺/Ca²⁺ exchanger (NCX), a 10-transmembrane domain protein mainly involved in the regulation of intracellular Ca²⁺ homeostasis, plays a crucial role in cerebral ischemia. In the present paper, we characterized the effect of the newly synthesized compound 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED) on the activity of the three NCX isoforms and on the evolution of cerebral ischemia. BED inhibited NCX isoform 3 (NCX3) activity (IC<inf>50</inf> = 1.9 nM) recorded with the help of single-cell microflorimetry, ⁴⁵Ca²⁺ radiotracer fluxes, and patch-clamp in whole-cell configuration. Furthermore, this drug displayed negligible effect on NCX2, the other isoform expressed within the CNS, and it failed to modulate the ubiquitously expressed NCX1 isoform. Concerning the molecular site of action, the use of chimera strategy and deletion mutagenesis showed that α1 and α2 repeats of NCX3 represented relevant molecular determinants for BED inhibitory action, whereas the intracellular regulatory f-loop was not involved. At 10 nM, BED worsened the damage induced by oxygen/glucose deprivation (OGD) followed by reoxygenation in cortical neurons through a dysregulation of [Ca²⁺]<inf>i</inf>. Furthermore, at the same concentration, BED significantly enhanced cell death in CA3 subregion of hippocampal organotypic slices exposed to OGD and aggravated infarct injury after transient middle cerebral artery occlusion in mice. These results showed that the newly synthesized 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride is one of the most potent inhibitor of NCX3 so far identified, representing an useful tool to dissect the role played by NCX3 in the control of Ca²⁺ homeostasis under physiological and pathological conditions.