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Topology Analysis of Drug Binding Sites on Serum Proteins using Photoaffinity Labeling Techniques

使用光亲和标记技术对血清蛋白上的药物结合位点进行拓扑分析

基本信息

批准号：
09470505
负责人：
OTAGIRI Masaki
金额：
$ 7.17万
依托单位：
KUMAMOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

Arylpropionic acid NSAIDs and benzodiazepines are known to bind to site II of human serum albumin (HAS). However, information on the amino acids of the binding site that interact with these drugs is still insufficient. Ketoprofen (KP), an arylpropionic acid NSAID possessing benzophenone moiety and flunitrazepam (FNZP), a benzodiazepine with nitrophenyl moiety were used as photoaffinity labeling agents to define the microenvironment of site II on HAS. CNBr fragments of the [ィイD114ィエD1C]KP photolabeled HAS of about 14 kDa and 9 kDa incorporated radioactivity. Competition experiment showed that the 14 kDa band was the specifically photolabeled band. The 14 kDa peptide was redigested with Achromobacter lyticus protease I (AP I) and then separated with capillary HPLC. The amino acid sequence of the [ィイD114ィエD1C]KP photolabeled peptide was concluded to be XCTESLVNRR, which corresponded to 476C - 500K of HAS. This region of HAS was reported to be part of the site II hydrophobic binding pocket. Benzodiazepine (BDZ) drugs are known to bind to both human serum albumin (HAS) and alphal-acid glycoprotein (AGP). It is generally accepted that benzodiazepine drugs bind to subdomain III A (site II) of HAS. CNBr fragment of about 20 kDa of the [ィイD13ィエD1H]FNZP photolabeled HAS incorporated most of the radioactivity while the major photolabeled band of AGP seemed to be the N-terminus CNBr fragment The 20 kDa band radioactivity decreased sharply when HAS was photolabeled with FNZP in the presence of myristic acid but not in the presence of diazepam. The reverse displacement effect was observed for the N-terminus band of AGP. Further fluorescent probe displacement experiment showed that FNZP failed to displace the site II probe dansylsarcosine. The key position in the BDZ molecular structure that governs the binding affinity to site II of HAS was concluded to be position 7 of ring A.

已知芳基丙酸NSAID和苯二氮卓类药物与人血清白蛋白（HAS）的位点II结合。然而，与这些药物相互作用的结合位点的氨基酸的信息仍然不足。以酮洛芬（KP）和氟硝西泮（FNZP）为光亲和标记物，研究了HAS表面II位点的微环境。掺入放射性的约14 kDa和9 kDa的[β-D114 β-D1C]KP光标记的HAS的CNBr片段。竞争实验表明，14 kDa条带为特异性光标记条带。14 kDa的肽用无色杆菌蛋白酶I（AP I）再消化，然后用毛细管HPLC分离。经光标记后的[β-D_（114）β-D_（1C）]KP的氨基酸序列为XCTESLVNRR，对应于HAS的476 C-500 K。据报道，HAS的该区域是位点II疏水结合口袋的一部分。已知苯二氮卓类（BDZ）药物可与人血清白蛋白（HAS）和唾液酸糖蛋白（AGP）结合。人们普遍认为苯二氮卓类药物与HAS的亚结构域III A（位点II）结合。[β-D_（13）β-D_（1H）]FNZP光标记的HAS的约20 kDa的CNBr片段掺入了大部分放射性，而AGP的主要光标记带似乎是N-末端CNBr片段。当HAS在肉豆蔻酸存在下而不是在地西泮存在下用FNZP光标记时，20 kDa带的放射性急剧下降。对AGP的N-末端条带观察到反向置换效应。进一步的荧光探针置换实验表明，FNZP不能置换位点II探针丹酰肌氨酸。BDZ分子结构中控制HAS位点II结合亲和力的关键位置被认为是A环的7位。