YM-244769, a Novel Na⁺/Ca²⁺Exchange Inhibitor That Preferentially Inhibits NCX3, Efficiently Protects against Hypoxia/Reoxygenation-Induced SH-SY5Y Neuronal Cell Damage

Abstract

We investigated the pharmacological properties and interaction domains of N- (3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide (YM-244769), a novel potent Na ϩ /Ca 2ϩ exchange (NCX) inhibitor, using various NCX-transfectants and neuronal and renal cell lines. YM-244769 preferentially inhibited intracellular Na ϩ -dependent 45 Ca 2ϩ uptake via NCX3 (IC 50 ϭ 18 nM); the inhibition was 3.8-to 5.3-fold greater than for the uptake via NCX1 or NCX2, but it did not significantly affect extracellular Na ϩ -dependent 45 Ca 2ϩ efflux via NCX isoforms. We searched for interaction domains with YM-244769 by NCX1/NCX3-chimeric analysis and determined that the ␣-2 region in NCX1 is mostly responsible for the differential drug response between NCX1 and NCX3. Further cysteine scanning mutagenesis in the ␣-2 region identified that the mutation at Gly833 markedly reduced sensitivity to YM-244769. Mutant exchangers that display either undetectable or accelerated Na ϩ -dependent inactivation, had markedly reduced sensitivity or hypersensitivity to YM-244769, respectively. YM-244769, like 2-[2-[4-(4-nitrobenzyloxyl)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), protected against hypoxia/reoxygenation-induced cell damage in neuronal SH-SY5Y cells, which express NCX1 and NCX3, more efficiently than that in renal LLC-PK 1 cells, which exclusively express NCX1, whereas 2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester (SN-6) suppressed renal cell damage to a greater degree than neuronal cell damage. These protective potencies consistently correlated well with their inhibitory efficacies for the Ca 2ϩ uptake via NCX isoforms existing in the corresponding cell lines. Antisense knockdown of NCX1 and NCX3 in SH-SY5Y cells confirmed that NCX3 contributes to the neuronal cell damage more than NCX1. Thus, YM-244769 is not only experimentally useful as a NCX inhibitor that preferentially inhibits NCX3, but also has therapeutic potential as a new neuroprotective drug.

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

“…Furthermore, it is well established that up to 1 M (dose used in the present study) does not affect the forward mode (37 The increased NCX3 reverse mode activity in MHS murine muscle cells suggest a compensatory mechanism to overcome the lower SR Ca 2ϩ loading described in MHS muscle cells (19,25,64) as a result of RyR1-MH leakiness (21). The reduced SR Ca 2ϩ levels are likely to promote an increased Ca 2ϩ influx through Orai and TRPC channels (19), and possibly other Ca 2ϩ channels (34).…”

“…Because KB-R7943 has several targets in the muscle cells, we tested YM-244769 that has high affinity for blocking the reverse mode of NCX3 (37). Exposure of fibers to YM-244769 (1 M) for 5 min did not modify [Ca 2ϩ ] r in MHN fibers (119 Ϯ 1 nM, Fig.…”

Molecular Pharmacology of the Amiloride Analog 3-Amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)-amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) as a Pan Inhibitor of the Na+-Ca2+Exchanger Isoforms NCX1, NCX2, and NCX3 in Stably Transfected Cells

“…In light of these pharmacological properties, CB-DMB can be considered a valid tool to inhibit, in those tissues that express all of the three NCX isoforms, the currents carried by the antiporter when it works in the forward or in the reverse mode of operation. The potency of CB-DMB is barely lower than that of the new isothiourea derivative YM-244769 which is the most potent available NCX1, NCX2, and NCX3 inhibitor working in the nanomolar range (Iwamoto and Kita, 2006). However, YM-244769 inhibits NCX only in the reverse mode of operation, whereas CB-DMB inhibited NCX when it acts bidirectionally.…”

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

“…In fact, most of drugs that inhibit NCX, including KB-R7943, YM-244769, and SN-6, display an activity in the micromolar range, whereas neurounina-1 exerts its effect on NCX1 and NCX2 in the picomolar and low nanomolar range of concentrations, with an EC 50 of 1 to 0.1 nM. Moreover, at variance with most NCX inhibitors (Iwamoto et al, 1996;Annunziato et al, 2004;Iwamoto and Kita, 2006), neurounina-1 did not display a significant difference in potency between the forward and reverse modes of -free medium or in the presence of the GABA uptake inhibitor SKF89976A. (E) effect of different concentrations of neurounina-1 on endogenous GABA release induced by 15 mM KCl (data were subtracted for basal release values).…”

“…Therefore, in recent years there has been great interest in the identification of new compounds capable of increasing NCX activity to limit the extension of ischemic brain damage ). To date, only NCX inhibitors are available including 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-N- [[(2,4-dimethylbenzyl) amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) , KB-R7943 (Watano et al, 1999), SEA0400 (Matsuda et al, 2001), SM-15811 (Hasegawa et al, 2003), SN-6 (Iwamoto et al, 2004a), and YM-244769 (Iwamoto and Kita, 2006). These inhibitors have a number of interesting features.…”