生物学杂志

生物学杂志 ›› 2026, Vol. 43 ›› Issue (5): 81-.doi: 10.3969/j.issn.2095-1736.2026.05.081

• 技术方法 • 上一篇    下一篇

CDC42活化态生物发光共振能量转移技术检测系统构建

周佳敏1， 林伟东2， 王雪飞2,3， 张　谦2， 崔云霞2， 赵明珠2， 张　健1,2
  

  1. 1. 南方医科大学 药学院， 广州 510515； 2. 上海交通大学 医学院， 上海 200025；
    3. 齐鲁制药集团药物研究院， 济南 250000
  • 出版日期:2026-10-18 发布日期:2026-10-20
  • 通讯作者: 张健，研究员，研究方向为分子变构机制，E-mail：jian.zhang@sjtu.edu.net；赵明珠，讲师，研究方向为变构分子改造与合成，E-mail：zhao_mingzhu@163.com；张健和赵明珠为共同通信作者
  • 作者简介:周佳敏，硕士研究生，研究方向为小分子药物合成，E-mail：xxxyogurt@163.com；林伟东，博士，研究方向为靶标蛋白筛选体系的构建，E-mail：lwdong21@163.com；周佳敏和林伟东为共同第一作者
  • 基金资助:
    海市高水平地方高校创新团队（SHSMU-ZDCX20212700）

#br# Development of a bioluminescence resonance energy transfer（BRET）-based assay system for detecting the activated state of CDC42 #br#

ZHOU Jiamin1, LIN Weidong2, WANG Xuefei2,3, ZHANG Qian2, CUI Yunxia2,ZHAO Mingzhu2, ZHANG Jian1,2#br#   

  1. 1. School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China;
    2. School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China;
    3. Pharmaceutical Research Institute, Qilu Pharmaceutical Group Co., Ltd., Jinan 250000, China
  • Online:2026-10-18 Published:2026-10-20

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摘要： CDC42 GTP酶在多种肿瘤中过表达，并通过激活促生长、血管生成及转移相关信号通路驱动肿瘤进展。为弥补细胞分裂周期蛋白CDC42激活状态检测方法的不足，本研究基于生物发光共振能量转移（BRET）技术，建立了一种高通量筛选体系，用于快速检测CDC42的激活状态。通过将CDC42与PAK1分别偶联海肾萤光素酶（RLUC）和增强型绿色荧光蛋白（EGFP），构建BRET检测系统。本研究系统优化蛋白融合方式（N端或C端融合RLUC/EGFP）及双质粒9种转染比例，发现CDC42-C-EGFP与PAK1-C-RLUC（9∶1）的组合可显著提升BRET信号，使BRET 信号比从初始的0.05提高至0.25，增强4倍，大幅提高了检测灵敏度。通过该体系，本研究验证了已知抑制剂（ML141、ARN25062、ARN24928）浓度依赖性的抑制效应，其中ARN25062显示最佳的抑制效果；同样ARN25062对肝癌细胞的生长、迁移以及侵袭表现出最佳抑制效果。表明该研究成功建立的BRET体系可有效表征CDC42抑制剂的能力。该体系为深入研究CDC42的调控机制及开发靶向药物提供了重要工具，为小G蛋白酶（如Rho家族）的功能研究及药物开发提供新策略。

关键词: 细胞周期分裂蛋白42, 小G蛋白, 生物发光共振能量转移, ARN25062, ML141

Abstract: CDC42 GTPase is overexpressed in multiple tumors and drives oncogenic progression by activating signaling pathways associated with cell proliferation, angiogenesis, and metastasis. To address the limitations of current methods for detecting cell division cycle 42 （CDC42）activation status, this study developed a high-throughput screening system based on bioluminescence resonance energy transfer （BRET）technology for rapid assessment of CDC42 activation. By coupling CDC42 with renilla luciferase （RLUC）and PAK1 with enhanced green fluorescent protein （EGFP）, a BRET detection system was constructed. Systematic optimization of protein fusion strategies （N-terminal or C-terminal fusion of RLUC/EGFP）and dual-plasmid transfection ratios （nine combinations）revealed that the combination of CDC42-C-EGFP with PAK1-C-RLUC （9∶1 ratio）significantly enhanced BRET signals, increasing the BRET ratio from 0.05 to 0.25 （4-fold increase）, thereby substantially improving detection sensitivity. Using this system, concentration-dependent inhibitory effects of known inhibitors （ML141, ARN25062, ARN24928）were validated. Among these, ARN25062 exhibited the strongest inhibitory potency and also demonstrated the strongest suppression of hepatocellular carcinoma cell proliferation, migration, and invasion, consistent with cell-based assay results. These findings confirmed that the established BRET system effectively characterized CDC42 inhibitor activity. The optimized BRET-based platform provided a critical tool for investigating CDC42 regulatory mechanisms and developing targeted therapeutics, while offering novel strategies for functional studies and drug discovery of small GTPases （e.g., Rho family proteins）.

Key words: CDC42, small G protein, BRET, ARN25062, ML141

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