STUDY ON CATALYTIC REACTION MECHANISM OF ADENOSYLHOMOCYSTEINE HYDRLASE AND SYNTHESIS OF THEIR MECANISM -BASED INHIBITORS

腺苷同型半胱氨酸水解酶催化反应机理的研究及其抑制剂的合成

• 批准号: 10672086
• 负责人: KITADE Yukio
• 金额: $ 2.05万
• 依托单位: GIFU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10672086/
• 关键词: SAH hydrolase; anti-viral agent; acyclonucleoside; recombinant enzyme; mechanism-based inhibitor; affinity-labeling probe; carbocyclic nucleoside; inhibitor of protein synthesis; 抗ウィルス薬; 遺伝子組換酵素

The cellular enzyme S-adenosyl-L-homocysteine (SAH) hydrolase (EC 3.3.1.1) has emerged as a target enzyme for the molecular design of anti-viral agents. During the course of studies on the preparation of biologically important nucleosides using the reductive cleavage of purine nucleosides, a DHPA analogue (FDHPA) possessing a formyl group at the 3'-position has been designed as a possible affinity-labeling probe for the elucidation of the catalytic site of SAH hydrolase. We describe a method for the preparation of a facile affinity-labeling probe and its biological properties against human recombinant SAH hydrolase.The cDNA for human SAH hydrolase has been cloned from human heparoma cell HepG2 by RT-PCR and the obtained human recombinant SAH hydrolase was purified according to the literature. Incubation with FDHPA caused inactivation of the human SAH hydrolase and the activity was not recovered by dialysis The values of KィイD2iィエD2 and kィイD2inactィエD2 were 8.8 μM and 0.09 minィイD1-1ィエD1, respectively. Taking the above results into consideration, it is deduced that FDHPA functions as a useful affinity-labeling probe of SAH hydrolase and provides a clue to elucidation of the molecular mechanism of SAH hydrolase aiming at the molecular design of anti-viral drugs.We also prepared several carbocyclic nucleosides and acyclic nucleosides for the discovery of now type SAH hydrolase inhibitors.

细胞酶S-腺苷-L-高半胱氨酸（SAH）水解酶（EC 3.3.1.1）已经成为抗病毒剂分子设计的靶酶。在利用嘌呤核苷还原裂解制备具有重要生物学意义的核苷的研究过程中，设计了一种在3 '-位上具有甲酰基的DHPA类似物（FDHPA），作为可能的亲和标记探针，用于阐明SAH水解酶的催化位点。我们用RT-PCR方法从人肝素瘤细胞HepG 2中克隆了人重组SAH水解酶的cDNA，并根据文献报道的方法纯化了重组SAH水解酶，制备了一种简便的人重组SAH水解酶亲和标记探针，并对其生物学特性进行了研究。与FDHPA孵育导致人SAH水解酶失活，透析不能恢复其活性。K_（10）D_（21）D_（22）和K_（10）D_（21）失活D_（22）值分别为8.8 μM和0.09 min D_（1 -1）D_（1 -1）D_（1 -1）。因此，FDHPA可以作为SAH水解酶的亲和标记探针，为进一步研究SAH水解酶的分子机制，设计抗病毒药物提供了线索，并制备了几种碳环核苷和非环核苷类化合物，为新型SAH水解酶抑制剂的发现奠定了基础。

项目成果

Yukio Kitade: "Synthesis of S-adenosyl-L-homocysteine hydrolase inhibitors and their biological activities"Nucleic Acids Symposium Series. 42. 25-26 (1999)

Yukio Kitade：“S-腺苷-L-同型半胱氨酸水解酶抑制剂的合成及其生物活性”核酸研讨会系列。

Kosaku Hirota: "A convenient Synthesis of acyclic adenosines with an unsaturated side chain by modification of 9-(2,3-0-isopropylidene-D-nbityl)adenine"Nucleosides & Nucleotides. 17. 1333-1345 (1998)

Kosaku Hirota：“通过修饰 9-(2,3-0-异亚丙基-D-nbityl)腺嘌呤，方便合成具有不饱和侧链的无环腺苷”