Molecular mechanisms of drug transport across cell membrane

药物跨细胞膜转运的分子机制

基本信息

批准号：
11694310
负责人：
ENDOU Hitoshi
金额：
$ 6.78万
依托单位：
Kyorin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

One of the remarkable features of the transporters responsible for the transmembrane transport of organic substances is their multispecificity in the substrate recognition, whose molecular mechanisms remain to be clarified. Even after the three-dimensional structures of the transporter proteins are solved by crystallography, it would be still difficult to understand the structural basis for the multispecific nature in the substrate recognition. Therefore, in the present investigation, we have performed structure-activity analysis using molecular pharmacological analysis.Neutral amino acid transporter LAT1 (L-type amino acid transporter 1) transports not only naturally-occurring L-amino acids nut also amino acid related drugs. In order to generate stably transected cell lines, we transected human LAT1 cDNA to S2 cells derived from mouse renal proximal tubule S2 segment of transgenic mice overexpressing SV40 large T antigen. We, then, examined the inhibitory effects of the compounds on the uptake of radiolabeled phenylalanine by the human LAT1-stable transfected cell line. We demonstrated that the phenylalanine uptake was inhibited by aromatic amino acid-related compounds such as L-dopa, alpha-methyldopa, triiodethyronine, thyroxine in a competitive manner. On the other hands phenylalanine-methylester, N-methylphenylalanine and dopamine had no effects on the LAT1-mediated transport. Computational analysis results indicated that presence of aromatic hydrophobic side chains as well as free carboxyl and free amino groups is essential to interacted with substrate binding site of LAT1.It was proved that the combination of the molecular pharmacological analysis and the cpmput ational analysis is the excellent means to reveal the structural basis of the substrate recognition of transporters.

负责有机物质跨膜转运的转运蛋白的显着特征之一是它们在底物识别中的多特殊性，其分子机制仍有待阐明。即使在通过晶体学求解转运蛋白的三维结构之后，仍然很难理解底物识别中多特异性性质的结构基础。因此，在本研究中，我们使用分子药理学分析进行了结构活性分析。中性氨基酸转运蛋白转运蛋白LAT1（L型氨基酸转运蛋白1）不仅运输自然存在的L-氨基酸NUT还与氨基酸相关的药物。为了产生稳定的细胞系，我们将人LAT1 cDNA转移到了源自过表达SV40大T抗原的转基因小鼠的小鼠肾近端小管S2段的S2细胞。然后，我们检查了化合物对人LAT1稳定转染细胞系对放射性标记苯丙氨酸摄取的抑制作用。我们证明了苯丙氨酸的摄取受到芳香氨基酸相关的化合物的抑制，例如L-DOPA，Alpha-Methyldopa，Triiodethyronine，Thyrosine，甲状腺素，甲状腺素的竞争方式。另一方面，苯丙氨酸 - 甲基酯，N-甲基苯胺和多巴胺对LAT1介导的转运没有影响。计算分析结果表明，芳香疏水侧链以及游离羧基和游离氨基组的存在对于与LAT1的底物结合位点相互作用至关重要。证明，这是分子药理分析和CPMPUP ATIANIal Analysis的组合是揭示转运剂的基质识别的结构基础的出色手段。