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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 (Mpro) and papain-like protease (PLpro), 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 Mpro by more than 60% at a concentration of 20 mu M, and four of them with high potency (IC50 < 10 mu M). 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 mu M and selectivity index values of 5.1, 3.1 and 6.5, respectively. The docking poses of MDAAs in SARS-CoV-2 Mpro 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., Journal of Natural Products, 86 (2), ISSN:0163-3864, ISSN:1520-6025

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a robust and powerful tool for studying biomacromolecules and their interactions. However, quantitative detection of high-mass analytes (kDa to MDa range) remains challenging for MALDI-MS. Herein, we successfully used commercially available purified proteins (β-galactosidase and BSA) as internal standards for high-mass MALDI-MS analysis and achieved absolute quantification of several high-mass analytes. We systematically evaluated four sample deposition methods, and using the sandwich deposition method with saturated sinapinic acid as the top layer, we performed a robust quantitative analysis by high-mass MALDI-MS. Combined with chemical cross-linking, this quantitative strategy was further used to evaluate the affinity of protein–protein interactions (PPIs), specifically of two soluble protein receptors (interleukin 1 receptor and interleukin 2 receptor) and two membrane protein receptors (rhodopsin and angiotensin 2 receptor 1) with their interaction partners. The measured dissociation constants of the protein complexes formed were between 10 nM and 5 μM. We expect this high-throughput, rapid method, which does not require labeling or immobilization of any of the interaction partners, to become a viable alternative to traditional biophysical methods for studying PPIs. ISSN:1520-6882 ISSN:0003-2700