Efficient discovery of potential inhibitors for SARS-CoV-2 3C-like protease from herbal extracts using a native MS-based affinity-selection method.

• A native mass spectrometry-based affinity-selection coupled with a natural compound identification procedure was developed. • With the optimized method, compounds that bind to SARS-CoV-2 3CLpro were screened out from complex mixtures. • Protein-ligand complexes and stoichiometric numbers could be visualized and alerts could be given for nonspecific binding. • The binding affinity between protein and compound could be easily determined by mass spectrometry. [Display omitted] A native MS-based affinity-selection method was developed to screen compounds that target SARS-CoV-2 3CLpro from TCMs crude extracts. The 3C-like protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the virus life cycle and is supposed to be a potential target for the treatment of coronaviral infection. Traditional Chinese medicines (TCMs) have played an impressive role in the treatment of COVID-19 in China. The effectiveness of TCM formulations prompts scientists to take continuous effort on searching for bioactive small molecules from the ancient resources. Herein, we developed a native mass spectrometry-based affinity-selection method for rapid screening of active small molecules from crude herbal extracts applied for COVID-19 therapy. Six common herbs named Lonicera japonica, Scutellaria baicalensis, Forsythia suspensa, Glycyrrhiza uralensis, Cirsium japonicum, and Andrographis paniculata were investigated. After preliminary separation of the crude extracts, the fractions were incubated with 3CLpro. A native MS-based affinity screening assay was then conducted to search for the protein-ligand complexes. A UHPLC-Q/TOF-MS with UNIFI data acquisition and data processing software was applied to identify the hit compounds. Standard compounds were used to verify the outcomes. Among the 16 hits, three flavonoids, baicalein, scutellarein and ganhuangenin, were identified as potential noncovalent inhibitors against 3CLpro with IC 50 values of 0.94, 3.02, and 0.84 μM, respectively. Their binding affinities were further characterized by native MS, with K d values being 1.43, 3.85, and 1.09 μM, respectively. Overall, we established an efficient native MS-based strategy for discovering 3CLpro ligands from crude mixtures, which supplies a potential strategy of small molecule lead discovery from TCMs. [ABSTRACT FROM AUTHOR]