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

Abstract

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 (IC₅₀ < 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 IC₅₀ 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 ¹H relaxation dispersion NMR spectroscopy.

Chart 1. Chemical Structures of Approved Drugs against SARS-CoV-2 (1–3), Reported Inhibitors of M^pro and PL^pro Used as Positive Controls (4–7), and Co-Crystallized Ligands of the Protein Structures Used as Queries (8–10)

Chart 2

Chart 3

Figure 1

Dose–response curves showing anti-SARS-CoV-2 activity of compounds 12 (A), 13 (B), and 15 (C) and of the Morus alba root bark extracts MA60 (D) and MA21 (E) in Calu-3 cells, as determined by a spike staining assay.

Figure 2

(A) Compound 8 (X77) bound to the butterfly-shaped substrate binding site of M^pro (PDB code: 6W63). (B–F) Docking poses of compounds 12 (green), 13 (purple), 14 (yellow), 15 (orange), and 16 (blue) in the binding site of 6W63. The substrate binding site is indicated as gray mesh. The water molecule is shown as a red sphere.

Figure 3

(A) STD NMR of M^pro and compound 16. The STD difference spectrum is shown in red, with the STD off spectrum (1D) superposed in blue. (B) Proposed binding epitope map of 16. (C) Combined presentation of the docking pose of 16 in 6W63. Determined interactions from STD NMR are highlighted in green (6B, 6C).