Flavonoids in Ampelopsis grossedentata as covalent inhibitors of SARS-CoV-2 3CLpro: Inhibition potentials, covalent binding sites and inhibitory mechanisms

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Abstract

Coronavirus 3C-like protease (3CLpro) is a crucial target for treating coronavirus diseases including COVID-19. Our preliminary screening showed that Ampelopsis grossedentata extract (AGE) displayed potent SARS-CoV-2-3CLpro inhibitory activity, but the key constituents with SARS-CoV-2-3CLpro inhibitory effect and their mechanisms were unrevealed. Herein, a practical strategy via integrating bioactivity-guided fractionation and purification, mass spectrometry-based peptide profiling and time-dependent biochemical assay, was applied to identify the crucial constituents in AGE and to uncover their inhibitory mechanisms. The results demonstrated that the flavonoid-rich fractions (10-17.5 min) displayed strong SARS-CoV-2-3CLpro inhibitory activities, while the constituents in these fractions were isolated and their SARS-CoV-2-3CLpro inhibitory activities were investigated. Among all isolated flavonoids, dihydromyricetin, isodihydromyricetin and myricetin strongly inhibited SARS-CoV-2 3CLpro in a time-dependent manner. Further investigations demonstrated that myricetin could covalently bind on SARS-CoV-2 3CLpro at Cys300 and Cys44, while dihydromyricetin and isodihydromyricetin covalently bound at Cys300. Covalent docking coupling with molecular dynamics simulations showed the detailed interactions between the orthoquinone form of myricetin and two covalent binding sites (surrounding Cys300 and Cys44) of SARS-CoV-2 3CLpro. Collectively, the flavonoids in AGE strongly and time-dependently inhibit SARS-CoV-2 3CLpro, while the newly identified SARS-CoV-2 3CLpro inhibitors in AGE offer promising lead compounds for developing novel antiviral agents.

Abbreviations

SARS-CoV-2
severe acute respiratory syndrome coronavirus 2
COVID-19
coronavirus disease 2019
3CLpro
Chymotrypsin-like protease
CoVs
coronaviruses
AGE
Ampelopsis grossedentata extract
IC50
half maximal inhibition concentration
EDTA
ethylene diamine tetraacetic acid
DTT
dithiothreitol
IAA
iodoacetamide
PMSF
phenylmethylsulfonyl fluoride
HEPES
2-[4-(2-hydroxyethyl) piperazin-1-yl] ethane sulfonic acid
HCl
hydrochloric acid
NaCl
sodium chloride
DMSO
dimethyl sulfoxide
PBS
potassium phosphate buffer
FRET
fluorescence resonance energy transfer
PDA
photo diode array
TOF-MS/MS
time of flight tandem mass spectrometry
nanoLC-MS/MS
nano liquid chromatography-tandem mass spectrometer
HCD
higher energy collision dissociation
MD
molecular dynamics
QFM
the orthoquinone form of myricetin
ACE2
angiotensin-converting enzyme 2
UGTs
UDP-glucuronosyltransferases

Keywords

SARS-CoV-2 3CLpro
Ampelopsis grossedentata extract
Covalent inhibitors

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1

Contributed equally.

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