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

Xiong, Yuan; Zhu, Guang-Hao; Zhang, Ya-Ni; Hu, Qing; Wang, Hao-Nan; Yu, Hao-Nan; Qin, Xiao-Ya; Guan, Xiao-Qing; Xiang, Yan-Wei; Tang, Hui; Ge, Guang-Bo

Coronavirus 3C-like protease (3CL(pro)) is a crucial target for treating coronavirus diseases including COVID-19. Our preliminary screening showed that Ampelopsis grossedentata extract (AGE) displayed potent SARS-CoV-2-3CL(pro) inhibitory activity, but the key constituents with SARS-CoV-2-3CL(pro) 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-2-3CL(pro) inhibitory activities, while the constituents in these fractions were isolated and their SARS-CoV-2-3CL(pro) inhibitory activities were investigated. Among all isolated flavonoids, dihydromyricetin, isodihydromyricetin and myricetin strongly inhibited SARS-CoV-2 3CL(pro) in a time-dependent manner. Further investigations demonstrated that myricetin could covalently bind on SARS-CoV-2 3CL(pro) 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 3CL(pro). Collectively, the flavonoids in AGE strongly and time-dependently inhibit SARS-CoV-2 3CL(pro), while the newly identified SARS-CoV-2 3CL(pro) inhibitors in AGE offer promising lead compounds for developing novel antiviral agents.