Citation: Chunyu Yan, Qinglong Qiao, Wei Zhou, Xuelian Zhou, Yonghui Chen, Lu Miao, Zhaochao Xu. FRET-based in vitro assay for rapid detecting of SARS-CoV-2 entry inhibitors[J]. Chinese Chemical Letters, ;2025, 36(5): 110258. doi: 10.1016/j.cclet.2024.110258 shu

FRET-based in vitro assay for rapid detecting of SARS-CoV-2 entry inhibitors

Chunyu Yan ^{a, b} ,
Qinglong Qiao ^a ,
Wei Zhou ^a ,
Xuelian Zhou ^{a, b} ,
Yonghui Chen ^a ,
Lu Miao ^{a, *,} ,
Zhaochao Xu ^{a, b, *,}

a.
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
b.
School of Chemistry, Dalian University of Technology, Dalian 116024, China

miaolu@dicp.ac.cn

zcxu@dicp.ac.cn

Received Date: 7 June 2024
Revised Date: 12 July 2024
Accepted Date: 15 July 2024
Available Online: 15 July 2024

Figures(5)

The continuous mutation and rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have led to the ineffectiveness of many antiviral drugs targeting the original strain. To keep pace with the virus' evolutionary speed, there is a crucial need for the development of rapid, cost-effective, and efficient inhibitor screening methods. In this study, we created a novel approach based on fluorescence resonance energy transfer (FRET) technology for in vitro detection of inhibitors targeting the interaction between the SARS-CoV-2 spike protein RBD (s-RBD) and the virus receptor angiotensin-converting enzyme 2 (ACE2). Utilizing crystallographic insights into the s-RBD/ACE2 interaction, we modified ACE2 by fusing SNAP tag to its N-terminus (resulting in SA740) and Halo tag to s-RBD’s C-terminus (producing R525H and R541H), thereby ensuring the proximity (< 10 nm) of labeled FRET dyes. We found that relative to the R541H fusion protein, R525H exhibited higher FRET efficiency, which attributed to the shortened distance between FRET dyes due to the truncation of s-RBD. Utilizing the sensitive FRET effect between SA740 and R525H, we evaluated its efficacy in detecting inhibitors of SARS-CoV-2 entry in solution and live cells. Ultimately, this FRET-based detection method was demonstrated high sensitivity, rapidity, and simplicity in solution and held promise for high-throughput screening of SARS-CoV-2 inhibitors.
- SARS-CoV-2,
- FRET,
- RBD-ACE2 interaction,
- Inhibitor,
- Fluorescence detection,
- Protein labeling
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沈阳化工大学材料科学与工程学院沈阳 110142