Inhibitor binding in the human renal low- and high-affinity Na+/glucose cotransporters
Inhibitor binding in the human renal low- and high-affinity Na+/glucose cotransporters
复制标题
DOI：
10.1124/jpet.107.129825
复制
发表时间：
2008-03-01
影响因子：
3.5
通讯作者：
Smith, Chari D.
中科院分区：
文献类型：
--
作者：
Pajor, Ana M.;Randolph, Kathleen M.;Smith, Chari D.
肾脏含有两种Na +/葡萄糖协同转运蛋白，称为SGLT 2和SGLT 1，沿近端小管长度沿着排列。低亲和力转运蛋白SGLT 2负责肾脏中大部分葡萄糖的重吸收。最近人们对SGLT 2作为使用基于苯基葡萄糖苷、根皮苷（根皮素-2 '-β-葡萄糖苷）的结构的选择性抑制剂治疗II型糖尿病的靶点感兴趣。在这项研究中，我们在表达hSGLT 1和hSGLT 2的COS-7细胞中检测了根皮苷和一种新的候选药物sergliflozin-A [（2-[4-甲氧基苯基]甲基）苯基β-D-吡喃葡萄糖苷]对α-甲基-D-吡喃葡萄糖转运的抑制作用。根皮苷的抑制是竞争性的，在hSGLT 1中的Ki值为0.3 μ M，在hSGLT 2中为39 nM。舍格列净-A的抑制作用也具有竞争性，hSGLT 1的Ki值为1 μ M，hSGLT 2的Ki值为20 nM。根皮素[3-（4-羟基苯基）-1-（2，4，6-三羟基苯基）-1-丙酮;根皮苷的苷元]是一种效力较低的抑制剂，在hSGLT 1中的IC 50值为142 μ M，在hSGLT 2中为25 μ M。被认为是在根皮苷结合位点的残基的定点诱变表明，只有Cys610参与抑制剂结合在人类转运蛋白。hSGLT 1中Cys610突变为赖氨酸导致所有抑制剂的IC50增加。相比之下，在hSGLT 2中类似Cys615的诱变产生相反的效果，根皮苷和sergliflozin-A的IC50降低。hSGLT 1和hSGLT 2之间突变效应的差异表明，这种半胱氨酸将关键残基保持在适当的位置，而不是直接参与抑制剂结合。
The kidney contains two Na+/glucose cotransporters, called SGLT2 and SGLT1, arranged in series along the length of the proximal tubule. The low-affinity transporter, SGLT2, is responsible for the reabsorption of most of the glucose in the kidney. There is recent interest in SGLT2 as a target for the treatment of type II diabetes using selective inhibitors based on the structure of the phenylglucoside, phlorizin (phloretin-2 '-beta-glucoside). In this study, we examined the inhibition of alpha-methyl-D-glucopyranose transport by phlorizin and a new candidate drug, sergliflozin-A [(2-[4-methoxyphenyl]methyl)phenyl beta-D-glucopyranoside], in COS-7 cells expressing hSGLT1 and hSGLT2. Inhibition by phlorizin was competitive, with K-i values of 0.3 mu M in hSGLT1 and 39 nM in hSGLT2. Inhibition by sergliflozin-A was also competitive, with K-i values of 1 mu M in hSGLT1 and 20 nM in hSGLT2. Phloretin [3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone; the aglucone of phlorizin] was a less potent inhibitor, with IC50 values of 142 mu M in hSGLT1 and 25 mu M in hSGLT2. Site-directed mutagenesis of residues believed to be in the phlorizin binding site showed that only Cys610 is involved in inhibitor binding in the human transporters. Mutation of Cys610 in hSGLT1 to lysine resulted in an increased IC50 for all inhibitors. In contrast, mutagenesis of the analogous Cys615 in hSGLT2 produced the opposite effect, a decrease in IC50 for phlorizin and sergliflozin-A. The differences in the effects of the mutations between hSGLT1 and hSGLT2 suggest that this cysteine holds key residues in place rather than participating directly in inhibitor binding.