Identification of Natural Products Inhibiting SARS-CoV-2 by Targeting Viral Proteases: A Combined in Silico and in Vitro Approach

[Image: see text] In this study, an integrated in silico–in vitro approach was employed to discover natural products (NPs) active against SARS-CoV-2. The two SARS-CoV-2 viral proteases, i.e., main protease (M(pro)) and papain-like protease (PL(pro)), were selected as targets for the in silico study. Virtual hits were obtained by docking more than 140,000 NPs and NP derivatives available in-house and from commercial sources, and 38 virtual hits were experimentally validated in vitro using two enzyme-based assays. Five inhibited the enzyme activity of SARS-CoV-2 M(pro) by more than 60% at a concentration of 20 μM, and four of them with high potency (IC(50) < 10 μM). These hit compounds were further evaluated for their antiviral activity against SARS-CoV-2 in Calu-3 cells. The results from the cell-based assay revealed three mulberry Diels–Alder-type adducts (MDAAs) from Morus alba with pronounced anti-SARS-CoV-2 activities. Sanggenons C (12), O (13), and G (15) showed IC(50) values of 4.6, 8.0, and 7.6 μM and selectivity index values of 5.1, 3.1 and 6.5, respectively. The docking poses of MDAAs in SARS-CoV-2 M(pro) proposed a butterfly-shaped binding conformation, which was supported by the results of saturation transfer difference NMR experiments and competitive (1)H relaxation dispersion NMR spectroscopy.