YM-244769, a novel Na+/Ca2+ exchange inhibitor that preferentially inhibits NCX3, efficiently protects against hypoxia/reoxygenation-induced SH-SY5Y neuronal cell damage.
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(+)/Ca(2+) exchange (NCX) inhibitor, using various NCX-transfectants and neuronal and renal cell lines. YM-244769 preferentially inhibited intracellular Na(+)-dependent (45)Ca(2+) uptake via NCX3 (IC(50) = 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(+)-dependent (45)Ca(2+) efflux via NCX isoforms. We searched for interaction domains with YM-244769 by NCX1/NCX3-chimeric analysis and determined that the alpha-2 region in NCX1 is mostly responsible for the differential drug response between NCX1 and NCX3. Further cysteine scanning mutagenesis in the alpha-2 region identified that the mutation at Gly833 markedly reduced sensitivity to YM-244769. Mutant exchangers that display either undetectable or accelerated Na(+)-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(2+) 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.
Iwamoto
Takahiro
T
Department of Pharmacology, School of Medicine, Fukuoka University, 7-45-1 Nanakuma Jonanku, Fukuoka 814-0180, Japan. tiwamoto@cis.fukuoka-u.ac.jp
Kita
Satomi
S
D000818:Animals; D001483:Base Sequence; D015687:Cell Hypoxia; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D017931:DNA Primers; D006801:Humans; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D015427:Reperfusion Injury / Q000517:prevention & control*; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors*
D017931:DNA Primers; C516554:N-(3-aminobenzyl)-6-(4-((3-fluorobenzyl)oxy)phenoxy)nicotinamide; C101832:SLC38A3 protein, human; D019831:Sodium-Calcium Exchanger; D009536:Niacinamide