A Biolayer Interferometry-Based SARS-COV-2 Mpro-Targeted Active Ingredients Recognition System: Construction and Application in Ligand Screening From Herbal Medicines

Drug discovery research targeting SARS-CoV-2 and other emerging pathogens remains critically important. Active compounds derived from plants frequently serve as lead compounds for further drug discovery; however, numerous unrelated chemical constituents in crude extracts may obscure the effective ingredients in LC-MS analysis. The aim of this study is to construct a biolayer interferometry (BLI)-based system for recognizing active ingredients that inhibit the main protease (Mpro) of SARS-CoV-2 and to identify the active chemical components binding to Mpro from herbal medicines. We developed a novel FRET fluorogenic probe by linking the amino acid sequences of the fluorescent proteins Lssmorange and mKate2 (Ls-mK). The interaction between traditional Chinese medicine and Mpro was analyzed using BLI. Ultrahigh performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was employed to analyze the composition of herbal medicines. Fluorescence detection and spectroscopy confirmed the successful construction of an Mpro inhibitor screening system. Lanqin Oral Liquid (LQL) and Gardeniae fructus exhibited strong inhibitory effects on Mpro. Ten compounds were identified from G. fructus extracts; among them, deacetyl asperulosidic acid methyl ester (DAAME) and Gardoside were found to strongly bind to Mpro, with dissociation constants (KD) of 3.41 μM and 801 nM, respectively. The half-maximal inhibitory concentrations (IC50) of DAAME and Gardoside for Mpro were 27.46 and 13.7 μM, respectively. This study established a functional Mpro inhibitor screening system. Among the 10 components identified from G. fructus that bind to Mpro, DAAME and Gardoside displayed strong binding and inhibitory activity, indicating their potential as lead compounds for inhibiting SARS-CoV-2 viral replication.