AR-C155858 is a potent inhibitor of monocarboxylate transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7-10.
AR-C155858 is a potent inhibitor of monocarboxylate transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7-10.
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DOI：
10.1042/bj20091515
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发表时间：
2010-01-15
期刊：
影响因子：
--
通讯作者：
Halestrap AP
中科院分区：
文献类型：
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作者：
Ovens MJ;Davies AJ;Wilson MC;Murray CM;Halestrap AP
在本研究中，我们表征了有效的MCT 1（单羧酸转运蛋白1）抑制剂AR-C155858的特性。使用大鼠红细胞中MCT 1的L-乳酸转运抑制剂滴定法测定MCT 1上AR-C155858结合位点（Et）的Ki值和数量以及转运蛋白的转换数（kcat）。推导值分别为2.3±1.4 nM、1.29±0.09 nmol/ml浓缩细胞和12.2±1.1 s-1。当在非洲爪蟾卵母细胞中表达时，MCT 1和MCT 2被AR-C155858强效抑制，而MCT 4则没有。对MCT 1的抑制显示出时间依赖性，并且该化合物在显微注射时也具有活性，这表明AR-C155858可能在与MCT 1上的细胞内位点结合之前进入细胞。结合MCT 1和MCT 4的不同结构域的几种嵌合转运蛋白的抑制剂敏感性的测量显示，AR-C155858的结合位点包含在MCT 1的C-末端一半内，并涉及TM（跨膜）结构域7-10。这与之前的数据一致，这些数据将Phe 360（TM 10中）和Asp 302加Arg 306（TM 8）确定为MCT 1与底物结合和易位的关键残基。对L-乳酸和丙酮酸的嵌合体的Km值的测量表明，分子的C-和N-末端两半影响转运动力学，这与我们提出的MCT 1的分子模型及其易位机制一致，该易位机制除了需要TM 8中的Asp 302和Arg 306之外还需要TM 1中的Lys 38 [Wilson，Meredith，Bunnun，Sessions和Halestrap（2009）J.Biol.Chem.284，20011-20021]。
In the present study we characterize the properties of the potent MCT1 (monocarboxylate transporter 1) inhibitor AR-C155858. Inhibitor titrations of L-lactate transport by MCT1 in rat erythrocytes were used to determine the Ki value and number of AR-C155858-binding sites (Et) on MCT1 and the turnover number of the transporter (kcat). Derived values were 2.3±1.4 nM, 1.29±0.09 nmol per ml of packed cells and 12.2±1.1 s−1 respectively. When expressed in Xenopus laevis oocytes, MCT1 and MCT2 were potently inhibited by AR-C155858, whereas MCT4 was not. Inhibition of MCT1 was shown to be time-dependent, and the compound was also active when microinjected, suggesting that AR-C155858 probably enters the cell before binding to an intracellular site on MCT1. Measurement of the inhibitor sensitivity of several chimaeric transporters combining different domains of MCT1 and MCT4 revealed that the binding site for AR-C155858 is contained within the C-terminal half of MCT1, and involves TM (transmembrane) domains 7–10. This is consistent with previous data identifying Phe360 (in TM10) and Asp302 plus Arg306 (TM8) as key residues in substrate binding and translocation by MCT1. Measurement of the Km values of the chimaeras for L-lactate and pyruvate demonstrate that both the C- and N-terminal halves of the molecule influence transport kinetics consistent with our proposed molecular model of MCT1 and its translocation mechanism that requires Lys38 in TM1 in addition to Asp302 and Arg306 in TM8 [Wilson, Meredith, Bunnun, Sessions and Halestrap (2009) J. Biol. Chem. 284, 20011–20021].