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.

The 5′-adenosine monophosphate-activated protein kinase (AMPK) is an important metabolic regulator. Its allosteric drug and metabolite binding (ADaM) site was identified as an attractive target for direct AMPK activation and holds promise as a novel mechanism for the treatment of metabolic diseases. With the exception of lusianthridin and salicylic acid, no natural product (NP) is reported so far to directly target the ADaM site. For the streamlined assessment of direct AMPK activators from the pool of NPs, an integrated workflow using in silico and in vitro methods was applied. Virtual screening combining a 3D shape-based approach and docking identified 21 NPs and NP-like molecules that could potentially activate AMPK. The compounds were purchased and tested in an in vitro AMPK α 1 β 1 γ 1 kinase assay. Two NP-like virtual hits were identified, which, at 30 µM concentration, caused a 1.65-fold (± 0.24) and a 1.58-fold (± 0.17) activation of AMPK, respectively. Intriguingly, using two different evaluation methods, we could not confirm the bioactivity of the supposed AMPK activator lusianthridin, which rebuts earlier reports.

A pigment-depleted extract from the heartwood of Pterocarpus santalinus L. f. (PS-DE) showed promising anti-SARS-CoV-2 activity with an IC50 of 29.9 μg/mL in Caco-2-F03 cells. To determine the potential active constituents within the extract prior to isolation, multi-informative molecular network (MN) was applied. Therefore, the extract was separated by high-performance counter-current chromatography (HPCCC) into 11 fractions which were subsequently tested for anti-SARS-CoV-2 activity and analysed by UPLC-tandem mass spectrometry (MS2). The resulting MN combines the bioactivity data of the fractions with the MS2 data. The MN analysis led to the targeted isolation of seven compounds including one pterocarpan (7) reported for the first time as constituent of P. santalinus and four so far undescribed natural products (NPs) that belong to the compound classes of arylpropanes (9), isoflavanones (10) coumestans (16) and 3-arylcoumarins (17), respectively. In total, 15 constituents from the heartwood of P. santalinus and one synthetic isoflavonoid that is structurally related to the natural metabolites were tested for anti-SARS-CoV-2 activity. Thereby, the two pterocarpans (−)-homopterocarpin (5) and (−)-medicarpin (2), the stilbene (E)-pterostilbene (1) and the isoflavonoid 7-O-methylgenistein (11) showed a distinct antiviral activity with IC50 values of 17.2, 33.4, 34.7, and 37.9 µM, respectively, and no cytotoxic effects against Caco-2-F03 cells (CC50 &gt; 100 µM). In addition, a structure-activity relationship (SAR) was proposed indicating structural requirements of pterocarpans for anti-SARS-CoV-2 activity. The herein presented results support the implementation of multi-informative molecular networks as powerful tool for dereplication and targeted isolation of bioactive NPs.

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