Identification of Natural Products Inhibiting SARS-CoV-2 by Targeting Viral Proteases: A Combined in Silico and in Vitro Approach.
Wasilewicz A, Kirchweger B, Bojkova D, Abi Saad MJ, Langeder J, Bütikofer M, Adelsberger S, Grienke U, Cinatl J, Petermann O, Scapozza L, Orts J, Kirchmair J, Rabenau HF, Rollinger JM.
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<sup>pro</sup>) and papain-like protease (PL<sup>pro</sup>), 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<sup>pro</sup> by more than 60% at a concentration of 20 μM, and four of them with high potency (IC<sub>50</sub> < 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 <i>Morus alba</i> with pronounced anti-SARS-CoV-2 activities. Sanggenons C (<b>12</b>), O (<b>13</b>), and G (<b>15</b>) showed IC<sub>50</sub> 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<sup>pro</sup> proposed a butterfly-shaped binding conformation, which was supported by the results of saturation transfer difference NMR experiments and competitive <sup>1</sup>H relaxation dispersion NMR spectroscopy.