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

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

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
Available online: 06 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

Electronic Supplementary Material

10.1016j.virs.2026.08.001-ESM.docx
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