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

Abstract

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 1.14, 3.04, 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 3 CLpro ligands from crude mixtures, which supplies a potential strategy of small molecule lead discovery from TCMs.

Phytovid19: a compilation of phytochemicals research in coronavirus

“…Another pentacyclinc triperpene Betulinic acid was reported to have activity against the same enzyme with IC50 10uM [ 59 ]. Andrographoside is a diterpenoid extracted from the traditional Chinese medicinal plant Andrographis paniculata having activity against SARS-CoV-2 [ 60 ]. Commiphora molmol (Engl.)…”

Affinity selection‐mass spectrometry in the discovery of anti‐SARS‐CoV‐2 compounds

“…Zhu et al ( 2022 ) applied native MS screening to the discovery of natural inhibitors of the SARS‐CoV‐2 protease 3CLpro. Reversed‐phase HPLC fractions of methanolic extracts of botanicals used in traditional Chinese medicine were screened for binding to recombinant 3CLpro (1 µM) in 10 mM ammonium acetate (pH 6.9) using an FTICR mass spectrometer.…”

“…In the search for therapeutic agents against COVID‐19, which is caused by the severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) (Figure 1 ), two AS‐MS approaches have been used to date, MagMASS (Muchiri et al, 2022 ; Pomplun et al, 2021 ) (Figure 2 ) and native MS screening (Littler, Liu, et al, 2021 ; Zhu et al, 2022 ) (Figure 3 ). Both approaches have been used in academic laboratories to find natural product inhibitors of SARS‐CoV‐2 infection and replication.…”

“…Any step of the SARS‐CoV‐2 virus infection and replication cycle is a potential target for antiviral intervention including cell entry, genome replication, viral maturation, or viral release (Figure 1 ). AS‐MS screening assays have been reported for the discovery of potential SARS‐CoV‐2 therapeutic agents targeting cell entry (Muchiri et al, 2022 ), genome replication (Littler, Liu, et al, 2021 ), and protein processing (Zhu et al, 2022 ).…”

“…Because these proteins are important for viral replication, inhibition of 3CLpro would inhibit SARS‐CoV‐2 replication and maturation. Native MS screening has been used to discover natural ligands of 3CLpro that inhibit proteolytic activity (Zhu et al, 2022 ).…”