YM-244769, a novel Na+/Ca2 exchange inhibitor that preferentially inhibits NCX3, efficiently protects against hypoxia/reoxygenation-induced SH-SY5Y neuronal cell damage
YM-244769, a novel Na+/Ca2 exchange inhibitor that preferentially inhibits NCX3, efficiently protects against hypoxia/reoxygenation-induced SH-SY5Y neuronal cell damage
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DOI：
10.1124/mol.106.028464
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发表时间：
2006-12-01
影响因子：
3.6
通讯作者：
Kita, Satomi
中科院分区：
文献类型：
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作者：
Iwamoto, Takahiro;Kita, Satomi
我们研究了N-（3-氨基苄基）-6-{4-[（3-氟苄基）氧基]苯氧基}烟酰胺（YM-244769）的药理学性质和相互作用域，其是一种新型有效的Na（+）/Ca（2+）交换（NCX）抑制剂，使用各种NCX转染子和神经元及肾细胞系。YM-244769优先抑制通过NCX 3的细胞内Na（+）依赖性（45）Ca（2+）摄取（IC（50）= 18 nM）;抑制作用是通过NCX 1或NCX 2摄取的3.8- 5.3倍，但它不显著影响通过NCX亚型的细胞外Na（+）依赖性（45）Ca（2+）流出。我们通过NCX 1/NCX 3嵌合分析搜索了与YM-244769的相互作用结构域，并确定NCX 1中的α-2区域主要负责NCX 1和NCX 3之间的差异药物反应。在α-2区域中的进一步半胱氨酸扫描诱变鉴定了Gly 833处的突变显著降低了对YM-244769的敏感性。显示不可检测或加速Na（+）依赖性失活的突变交换剂对YM-244769的敏感性或超敏性分别显著降低。YM-244769，如2-[2-[4-（4-硝基苄氧基）苯基]乙基]异噻唑烷甲磺酸盐（KB-R7943）在表达NCX 1和NCX 3的神经元SH-SY 5 Y细胞中比在仅表达NCX 1的肾LLC-PK（1）细胞中更有效地保护免受缺氧/再氧合诱导的细胞损伤，而2-[ 4-（4-硝基苄氧基）苄基]噻唑烷-4-羧酸乙酯（SN-6）抑制肾细胞损伤的程度大于神经元细胞损伤。这些保护效力与其通过存在于相应细胞系中的NCX异构体对Ca（2+）摄取的抑制效力一致地良好相关。在SH-SY 5 Y细胞中反义敲除NCX 1和NCX 3证实了NCX 3比NCX 1更有助于神经元细胞损伤。因此，YM-244769不仅在实验上可用作优先抑制NCX 3的NCX抑制剂，而且还具有作为新的神经保护药物的治疗潜力。
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/reoxygenationinduced 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.