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Updated: Aug 17, 2026
Fluorescence-Based Detection of FEN1 Nuclease Activity and Screening of Small-Molecule Inhibitors
Published on: June 27, 2025
An AIE fluorescence sensor enables the discovery of Cathepsin L inhibitors in Xuebijing injection
Shijia Liu1, Wenxin Zhang2, Jiaoli Lin3
1State Key Laboratory on Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
Abstract:
Cathepsin L (CTSL), a lysosomal cysteine protease implicated in various human pathological processes, including cancer and COVID-19, represents a promising therapeutic target. Herein, we present a simple and selective aggregation-induced emission (AIE)-based fluorescent sensor for CTSL inhibitor screening. The sensor was constructed by linking an AIE fluorophore with a CTSL-specific substrate peptide and comprehensively characterized using spectroscopic, microscopic, and microplate assay techniques. CTSL-mediated hydrolysis triggered fluorescence quenching via fluorophore deaggregation, whereas inhibitor presence restored the signal, enabling sensitive and quantitative monitoring of CTSL activity with a linear response across the tested range. Furthermore, coupling this platform with ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry allowed identification of the major CTSL-inhibitory constituents in Xuebijing (XBJ) injection, including protocatechuic acid, oxypaeoniflorin, paeoniflorin, and senkyunolide I. This study establishes a rapid and effective approach for uncovering bioactive compounds from traditional Chinese medicine that regulate viral entry mechanisms.
Insights
We developed a novel fluorescent sensor to screen for Cathepsin L (CTSL) inhibitors, crucial for diseases like cancer and COVID-19. This sensor successfully identified active compounds in traditional Chinese medicine, aiding drug discovery.
Area of Science:
- Biochemistry
- Chemical Biology
- Pharmacology
Background:
- Cathepsin L (CTSL) is a lysosomal protease involved in cancer and viral infections, making it a key therapeutic target.
- Developing selective inhibitors for CTSL is crucial for treating various human diseases.
Purpose of the Study:
- To create a simple and selective aggregation-induced emission (AIE)-based fluorescent sensor for screening CTSL inhibitors.
- To identify bioactive compounds from traditional Chinese medicine (TCM) that inhibit CTSL activity.
Main Methods:
- Constructed an AIE fluorescent sensor by conjugating an AIE fluorophore with a CTSL-specific substrate peptide.
- Characterized the sensor using spectroscopic, microscopic, and microplate assay techniques.
- Utilized ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) for compound identification.
Main Results:
- The AIE sensor demonstrated sensitive and quantitative monitoring of CTSL activity through fluorescence changes.
- CTSL-mediated hydrolysis led to fluorescence quenching, which was restored upon inhibitor presence.
- Identified four major CTSL-inhibitory compounds in Xuebijing (XBJ) injection: protocatechuic acid, oxypaeoniflorin, paeoniflorin, and senkyunolide I.
Conclusions:
- The developed AIE fluorescent sensor provides a rapid and effective platform for CTSL inhibitor screening.
- This approach facilitates the discovery of novel bioactive compounds from TCM that modulate viral entry mechanisms.
- The identified compounds from XBJ injection show potential for therapeutic applications targeting CTSL-related diseases.
