An AIE fluorescence sensor enables the discovery of Cathepsin L inhibitors in Xuebijing injection.

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.