基础医学与临床 ›› 2022, Vol. 42 ›› Issue (6): 933-939.doi: 10.16352/j.issn.1001-6325.2022.06.025

• 研究论文 • 上一篇    下一篇

高通量筛选NMNAT1抑制剂及其肿瘤杀伤作用

沙务嘎, 李隽*   

  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
  • 收稿日期:2022-03-23 修回日期:2022-04-22 出版日期:2022-06-05 发布日期:2022-06-02
  • 通讯作者: * jun_li@ibms.pumc.edu.cn
  • 基金资助:
    中国医学科学院医学与健康科技创新工程(CIFMS2021-I2M-1-050)

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

SHA Wu-ga, LI Jun*   

  1. Department of Biochemistry and Molecular Biology,Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC,Beijing 100005,China
  • Received:2022-03-23 Revised:2022-04-22 Online:2022-06-05 Published:2022-06-02
  • Contact: * jun_li@ibms.pumc.edu.cn

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2 280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。

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

Abstract: 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 2 280 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.

Key words: nicotinamide single nucleotide adenosine transferase 1(NMNAT1), protein purification, high-throughput chemical screening, apoptosis

中图分类号: