Abstract

Target‐based high‐throughput screening (HTS) is an efficient way to identify potent drugs. However, the accuracy of HTS could be affected by Pan‐Assay Interference Compounds (PAINS). One reason for the generation of PAINS is that the inherent photophysical property of screened compounds could interfere with typically used assay signals including absorption and fluorescence. Our previous studies indicate that the fluorescent probe based on the fluorophore with characteristics of aggregation‐induced emission (AIE) could provide high accuracy of HTS, especially for the fluorescent natural products. Herein, we report an AIE‐based fluorescent probe for the main protease (Mpro) of SARS‐CoV‐2. We designed and synthesized an AIE fluorescent probe ZLHG5, which has a site that can be specifically cleaved by Mpro to produce a light‐up fluorescence. Thanks to the large Stokes shift of AIE fluorophore (~300 nm), the probe could be effectively used for HTS of Mpro inhibitors. After screening a library of fluorescent natural products with ZLHG5, we obtained two coumarin‐originated natural compounds with potent inhibitory activity towards Mpro protease. This study provides both useful fluorescent HTS probe and potent inhibitors for Mpro protease.