高通量筛选NMNAT1抑制剂及其肿瘤杀伤作用  

High-throughput screening of inhibitors of NMNAT1 and their tumor-targeting functions

作　　者：沙务嘎 李隽 SHA Wu-ga;LI Jun(Department of Biochemistry and Molecular Biology,Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC,Beijing 100005,China)

机构地区：[1]中国医学科学院基础医学研究所,北京协和医学院基础学院生物化学与分子生物学系,北京100005

出　　处：《基础医学与临床》2022年第6期933-939,共7页Basic and Clinical Medicine

基　　金：中国医学科学院医学与健康科技创新工程(CIFMS2021-I2M-1-050)。

摘　　要：目的利用在大肠杆菌体系中诱导表达的人源NMNAT1蛋白筛选其抑制剂,寻找治疗肿瘤的新药物。方法利用酶切连接体系将人源NMNAT1插入pET21b(+)载体,将重组质粒转化入大肠杆菌BL21(DE3)菌株过表达蛋白,对重组蛋白进行纯化,通过3段式酶联荧光法在2280种天然化合物中筛选具有抑制NMNAT1活性的化合物,并在细胞水平验证其对肿瘤细胞的杀伤效果。结果纯化得到较高纯度和活性的人重组NMNAT1蛋白;筛选出6种高效的NMNAT1抑制剂;其中一种抑制剂fraxetin能降低乳腺癌细胞NAD+含量,促进细胞凋亡,与敲低NMNAT1后转录组测序(RNA-seq)分析得出的细胞凋亡通路增强一致。结论Fraxetin是NMNAT1强效抑制剂,能够促进乳腺癌细胞凋亡。Objective To find new drug candidates for cancer treatment by screening inhibitors of NMNAT1 using recombinant human NMNAT1 expressed in E.coli system.Methods The human NMNAT1 was inserted into the pET21b(+)vector and the recombinant plasmid was transformed into E.coli BL21(DE3)strain for over expression of NMNAT1 protein.The enzymatic activity of NMNAT1 was measured by a three-step coupled fluorescence assay,and 2280 chemicals from a nature compounds library were screened using this assay for potential inhibitors of NMNAT1.The potential target compounds that inhibited NMNAT1 were verified at the cellular level and tested for tumor-killing effect.Results The recombinant human NMNAT1 proteins with high purity and activity were obtained and 6 kinds of high-efficiency NMNAT1 inhibitors were identified.One of the them,fraxetin,efficiently reduced the NAD+content of breast cancer cells and promoted cell apoptosis.RNA-seq analysis of NMNAT1 knock-down cells revealed the function to enhance apoptosis which is consistent with the cellular effect of NMNAT1 inhibitor.Conclusions Fraxetin is a high-efficiency NMNAT1 inhibitor to promote apoptosis of breast cancer cells.

关 键 词：烟酰胺单核苷酸腺苷转移酶1(NMNAT1) 蛋白纯化 高通量药物筛选 凋亡 

分 类 号：R34[医药卫生—基础医学]