A modular APEX biosensor enables sensitive and visualized monitoring of viral protease activity

Biao Li; Bao Dong; Yuehong Chen; Jing Li; Xiaoping Kang; Yuchang Li; Sen Zhang; Jiaqi Xiong; Zhengwei Zhang; Jun Hu; Shuimeng Song; Shenghai Huang; Ye Feng; Tao Jiang

doi:10.1016/j.virs.2026.08.001

August 2026

. doi: 10.1016/j.virs.2026.08.001

Citation: Biao Li, Bao Dong, Yuehong Chen, Jing Li, Xiaoping Kang, Yuchang Li, Sen Zhang, Jiaqi Xiong, Zhengwei Zhang, Jun Hu, Shuimeng Song, Shenghai Huang, Ye Feng, Tao Jiang. A modular APEX biosensor enables sensitive and visualized monitoring of viral protease activity .VIROLOGICA SINICA, 2026, 41(4) : 972-987. http://dx.doi.org/10.1016/j.virs.2026.08.001

一种模块化APEX生物传感器可实现病毒蛋白酶活性的灵敏可视化检测

李标 ^a,b ,
董豹 ^a,b ,
陈月红 ^b ,
李靖 ^b ,
康晓平 ^b ,
李裕昌 ^b ,
张森 ^b ,
熊佳祺 ^c ,
张正伟 ^a ,
胡军 ^a ,
宋水萌 ^c ,
黄升海 ^a,, ,
冯烨 ^b,, ,
姜涛 ^a,b,c,,

a 安徽医科大学基础医学院;
b 军事医学科学院病原微生物与生物安全国家重点实验室;
c 牡丹江医学院公共卫生学院

通讯作者： 黄升海, shhuang@ahmu.edu.cn; 冯烨, fengye621@126.com; 姜涛, jiang_tao@126.com
收稿日期： 2026-03-12
录用日期： 2026-08-03

摘要

病毒蛋白酶是广谱抗病毒药物研发的核心靶点，亟需一套可在贴近生理状态的细胞环境中精准评估抑制剂活性的筛选平台。传统检测方法（如荧光蛋白检测、荧光共振能量转移 FRET 技术）存在明显缺陷：易受化合物非特异性构象干扰，且无法真实反映胞内抑制效果。为此，本研究构建了两种基于抗坏血酸过氧化物酶（APEX）的模块化生物传感器 TS3AR 与 C3SIR。其检测机制如下：蛋白酶特异性切割底物识别序列后，可促使拆分型 APEX 片段重新组装、恢复酶催化活性，并通过级联放大反应产生荧光信号。以冠状病毒主蛋白酶（Mpro）为模型开展验证实验，结果显示 TS3AR 传感器可将酶活检测信噪比提升至 1500 倍；C3SIR 传感器则能有效规避传统方法中由构象干扰引发的假阳性，精准筛选出恩司特韦、PF-00835231、奈玛特韦等高效 Mpro 抑制剂。同时，仅替换蛋白酶识别序列，该模块化传感器便可灵活检测多种冠状病毒（新冠病毒、中东呼吸综合征病毒等）的主蛋白酶（Mpro），同时适用于肠道病毒71型、EB病毒、甲肝病毒等其他病毒蛋白酶的活性检测与药物筛选评价。本研究由此为新发及现有病毒的广谱抗病毒药物快速研发，提供了一种稳定、特异性强的胞内药物筛选工具。
- 病毒蛋白酶
- , 冠状病毒（CoV）
- , 生物传感器
- , 抗坏血酸过氧化物酶（APEX）

A modular APEX biosensor enables sensitive and visualized monitoring of viral protease activity

Biao Li ^a,b ,
Bao Dong ^a,b ,
Yuehong Chen ^b ,
Jing Li ^b ,
Xiaoping Kang ^b ,
Yuchang Li ^b ,
Sen Zhang ^b ,
Jiaqi Xiong ^c ,
Zhengwei Zhang ^a ,
Jun Hu ^a ,
Shuimeng Song ^c ,
Shenghai Huang ^a,, ,
Ye Feng ^b,, ,
Tao Jiang ^a,b,c,,

a. School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China;
b. State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China;
c. School of Public Health, Mudanjiang Medical University, Mudanjiang 157011, China

Corresponding author: Shenghai Huang, shhuang@ahmu.edu.cn Ye Feng, fengye621@126.com Tao Jiang, jiang_tao@126.com
Received Date: 12 March 2026
Accepted Date: 03 August 2026

Abstract

Viral proteases are key targets for the development of broad-spectrum antiviral drugs development. However, screening platform capable of accurately assessing inhibitor activity within physiologically relevant cellular environments remain urgently needed. Traditional methods, such as fluorescent protein assays and Förster resonance energy transfer (FRET), suffer from significant limitations, including susceptibility to non-specific conformational interference by test compounds and an inability to faithfully reflect intracellular inhibitory effects. To address these challenges, we constructed two modular biosensors (TS3AR and C3SIR) based on engineered ascorbate peroxidase (APEX). Their detection mechanism relies on specific cleavage of the substrate recognition sequence by the target protease, which triggers the reassembly of split APEX fragments, restores enzymatic activity, and generates fluorescent signals generated via cascade amplification reaction. Validation using the coronavirus main protease (Mpro) as a model showed that the TS3AR sensor achieved the signal-to-noise ratio up to 1500-fold for enzyme activity detection, while the C3SIR sensor effectively avoided the false positives caused by conformational interference seen in traditional methods and accurately identified high-potency Mpro inhibitors, including enstrelvir, PF-00835231, and nirmatrelvir. Moreover, by replacing the protease recognition sequence, these modular biosensors can be flexibly adapted for activity analysis and drug evaluation of Mpro from various coronaviruses (e.g., SARS-CoV-2, MERS-CoV) as well as other viral proteases (e.g., enterovirus 71, Epstein-Barr virus and hepatitis A virus). Overall, this platform provides a reliable, highly specific intracellular screening tool to accelerate the development of broad-spectrum therapeutics against both emerging and existing viral threats.
- Viral protease
- , Coronavirus (CoV)
- , Biosensor
- , Ascorbate peroxidase

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10.1016j.virs.2026.08.001-ESM.docx