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Development of SARS virus inhibitor using unusual amino acid-containing natural products as seeds

以含有罕见氨基酸的天然产物为种子开发SARS病毒抑制剂

基本信息

批准号：
18590010
负责人：
AKAJI Kenichi
金额：
$ 2.44万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590010/
关键词：
SARS 3CL-protease inhibitor miraziridine A aziridine Tokaramide A

项目摘要

The 3C-like (3CL) protease, a thiol protease, of severe acute respiratory syndrome (SARS) coronavirus is a key enzyme for the maturation of SARS coronavirus. In the production of mature 3CL protease from the corresponding MBP (maltose binding protein)-His (six histidine)-Flag tag-fused protein, we identified two fragment proteins derived from degradation of the mature 3CL protease, and found for the first time that mature SARS 3CL protease is subject to degradation at the 188Arg/189Gln site, resulting in loss of catalytic activity. Mutation of Arg at the 188 position to Ile remarkably increased the stability of the protease, and the resulting R188I mutant protease could digest the conserved undecapeptide substrate with the efficiency of K_m = 33.8μM and k_<cat> = 4753 s^<-1>. Addition of His tag to the C-terminus of the mutant protease decreased the catalytic activity to 0.03 of the parent protease.We then achieved syntheses of natural products containing unusual amino acids to search seeds compounds those can act as SARS 3CL-protease inhibitor. As a suitable candidate, compound, we selected miraziridine A, a pentapeptide derivative isolated from marine sponge, and its truncated analogs. To construct the backbone of miraziridine A, a side-chain-unprotected vinylogous arginine was condensed with an aziridine-containingfragment prepared by a conventional solid-phase procedure. An analog lacking the vinylogous arginine site showed comparable inhibitoryactivity with miraziridine A, whereas an analog lacking the aziridine site showed remarkably weak inhibitory activity for cathepsin B, a typical thiol protease.We also find that a substrate-based tetra-peptide aldehyde, Ac-Ala-Val-Leu-NHCH (CH_2CH_2CON(CH_3)_2)-CHO, moderately inhibited the catalytic activity of the R188I mutant 3CL protease, whereas E-64, a typical cysteine protease inhibitor, showed no inhibitory activity for the mutant 3CL protease.

严重急性呼吸综合征(SARS)冠状病毒的3C-样(3CL)蛋白水解酶是SARS冠状病毒成熟的关键酶，是一种硫醇蛋白水解酶。在从相应的MBP(麦芽糖结合蛋白)-His(六组氨酸)-Flag Tag-融合蛋白生产成熟3CL蛋白酶的过程中，我们鉴定了两个来自成熟3CL蛋白酶降解的片段蛋白，并首次发现成熟的SARS 3CL蛋白酶在188Arg/189Gln位点发生降解，导致催化活性丧失。突变后的R188I酶能消化保守的十一肽底物，其消化效率分别为K_m=33.8μM和k_&lt；CAT&gt；=4753 S^&l；-1&gt；将His标签添加到突变蛋白的C末端，使其催化活性降至亲本蛋白的0.03。然后，我们合成了含有特殊氨基酸的天然产物，以寻找可以作为SARS 3CL-蛋白酶抑制剂的种子化合物。作为一个合适的候选化合物，我们选择了从海绵中分离出来的五肽衍生物咪嗪甲素及其截短类似物。为了构建咪嗪甲素的主链，用传统的固相法将侧链不受保护的乙烯精氨酸与含有氮杂环丙烷的片段缩合。缺少精氨酸基团的类似物对典型的硫醇蛋白水解酶组织蛋白B的抑制活性与咪嗪A相当，而缺乏氮杂环的类似物对组织蛋白酶B的抑制活性非常弱。我们还发现底物四肽醛Ac-Ala-Val-Leu-NHCH(CH_2CH_2CON(CH_3)_2)-CHO对R188I突变的3CL蛋白酶的催化活性有适度的抑制作用，而典型的半胱氨酸蛋白酶抑制剂E-对突变的3CL蛋白酶没有抑制活性。

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Evaluation of substrate specificity and inhibition at PR/p3 cleavage site of HTLV-1 protease

HTLV-1 蛋白酶 PR/p3 切割位点的底物特异性和抑制评价

DOI：
发表时间：
2006
期刊：
Bioorg. Med. Chem. Lett. 16
影响因子：
0
作者：
Mi hara;Y.; Ojima;H.; Imahori;T.; Yoshimura;Y.; Ouchi;H.; Takahata;H.;Hiromi Naka
通讯作者：
Hiromi Naka

Synthetic studies on miraziridine A, a cysteine protease inhibitor isolated from Theonella aff. mirabilis

米拉西啶 A 的合成研究，一种从 Theonella aff 中分离的半胱氨酸蛋白酶抑制剂。

DOI：
发表时间：
2006
期刊：
Peptide Science 2006
影响因子：
0
作者：
Mi hara;Y.; Ojima;H.; Imahori;T.; Yoshimura;Y.; Ouchi;H.; Takahata;H.;Hiromi Naka;Hiroyuki Konno
通讯作者：
Hiroyuki Konno

Studies on Substrate Specificity at PR/p3 Cleavage Site of HTVL-1 Protease

HTVL-1蛋白酶PR/p3切割位点底物特异性的研究