2.1. Virtual Screening
Computational approach comprising virtual screening, molecular docking and molecular dynamics (MD) simulation is a widely used method for the exploration of novel inhibitors against a target protein [13, 14], and cross docking is an approach to find the best structures for protein have been used in many studies [15-17]. Thus, the best crystal structure was selected for further study. In this paper, the crystal structures of ACE2 were obtained from RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) and reported in the literatures [2,4,18]. DiscoveryStudio 4.0 software was used to the preparation of ligand and receptor. A comparison of the structures of ACE2 (2019-nCoV-RBD-ACE2) and 1R4L indicated that ACE2 (2019-nCoV-RBD-ACE2)  was most similarity with 1R4L (see Supplementary data Figure S1). The active site was defined by ligand of ACE2 complex (1R4L). Ligand (MLN-4760) was docked in the active site of ACE2 (2019-nCoV-RBD-ACE2) and ACE2 (1R4L) (Figure 1C). Best docking pose was output on the basis of glide score (best docking energy), root-mean-square deviation (RMSD) and docking binding affinity (Ki) were calculated between the bioactive and docking conformations (Table 1). As shown in Table 1, RMSD values were less 0.3 nm and Ki values were same order of magnitude as reported biological activity. It was suggested that ACE2 (2019-nCoV-RBD-ACE2) was the most suitable one.
Virtual screening represents a fast approach to identify novel hit structures and has been widely employed in modern drug discovery campaign. In our efforts to identify novel ACE2 inhibitors, we virtually screened (see Supplementary data Table S1.) 565 compounds from 101 kinds of Chinese medicinal and edible plants utilizing AutoDock Vina (v.1.0.2.) . By applying a docking score cutoff of ≥ 9.0 kcal/mol, we identified 33 compounds (~5%) with the maximum scores (Table 2). These compounds were used for further assessing the physiochemical.
2.2. Physiochemical Properties
To further screen anti-2019-nCoV agents from 33 compounds (~5%), the physiochemical properties of the identified compounds were calculated by MarvinSketch version 5.3.8 , including molecular weight (MW), molecular surface area (MSA), polar surface area (PSA), relative polar surface area (%PSA), calculated partition coefficient (ClogP), calculated distribution coefficient at pH 7.4 (ClogD7.4), hydrogen bond donor (HD), hydrogen bond acceptor (HA), and rotatable bond (RB) (Table 3). For compounds (ZN00013, ZN00061, ZN00070, ZN00325, ZN00339, ZN00378, ZN00441 and ZN00451) had MW ranges between 426–821 g/mol, PSA were with the 9.23–256.0 Å2 range, ClogP < 5, ClogD7.4 < 5, with 1–7 HD and RB ≈ 10. Thus, 8 identified compounds matched Lipinski’s rule [21, 22], which were selected for activity test.
2.3. ACE2 kinase Inhibitory Assay
To assess the inhibitory potency of the identified compounds, eight compounds were subjected to in vitro ACE2 kinase inhibitory assay using Fluorescence assay method (see Supplementary data Supporting Text) [7, 23]. The inhibition rate of those compounds was presented in Table 2. Interestingly, ZN00061 (monoammonium glycyrrhizinate), ZN00070 (glycyrrhizic acid methyl ester), ZN00441 (ginsenoside Rg6) and ZN00451 (ginsenoside F1) shown the most potent activity against ACE2 kinase among eight compounds. Ginsenoside Rg6 (inhibition rate: 81.62%) and ginsenoside F1 (inhibition rate: 60.70%) were isolated from ginseng [24, 25]. Ginseng is one of the most commonly used traditional medicines in China, korea, Japan, and other Asian countries, shows various biological effects including anticancer, antioxidative, antiaging, neurovascular modulatory, antiviral, and other activities [26-29]. Ginsenoside Rg6 and ginsenoside F1 show antitumor, antioxidant and anti-inflammatory effects in vitro [30-33]. Previous study has reported that ginsenoside Rb1 exerted inhibitory activity against ACE . This study could provide evidence for the biological function of gnsenosides at the molecular level. Monoammonium glycyrrhizinate (inhibition rate: 51.68%) and glycyrrhizic acid methyl ester (inhibition rate: 50.40%) were isolated from Glycyrrhiza uralensis Fisch (Glycyrrhiza uralensis F.) [35, 36]. Glycyrrhiza uralensis F., an ancient herbal medicine in traditional Chinese medicine, has been used for thousands of years. It has several important pharmacological activities, including anti-oxidant, anti-cancer, anti-inflammatory, anti-ulcer, anti-viral, and anti-HIV [37-39]. Monoammonium glycyrrhizinate and glycyrrhizic acid methyl ester have not been reported in the treatment of ACE2 related diseases. However, the results showed that two compounds had a certain inhibitory effect on ACE2 in vitro and could be used as a lead compounds for further study.
2.4. Molecular Docking
To further investigate the potential binding between ACE2 and the compounds, the molecular docking was performed. The complex structures for monoammonium glycyrrhizinate, glycyrrhizic acid methyl ester, ginsenoside Rg6 and ginsenoside F1 were presented in Figure 2. The key residues were labeled and the important molecular interaction including the hydrogen bonds (black dotted lines), hydrophobic interactions (red dotted lines), and salt bridges (yellow dotted lines) were summarized in Table 4. As shown in Figure 2A, it was observed that the complex was stabilized by three hydrogen bonds (with ASN-63 and TYR-510), two hydrophobic interactions (with TYR-50 and PHE-504) and three salt bridges (with ASN-273 and HIS-345). The docking binding energy (∆G) of the interaction and the corresponding docking binding affinity (Kd) were estimated to be -1.63 kcal/mol and 6.23 × 102 M-1, respectively. We found that glycyrrhizic acid methyl ester binds at the site of ACE2 by forming five hydrogen bonds (with SER-124, TYR-199, TRP-203, ASP-509 and TYR-510), two hydrophobic interactions with THR-125 and ASP-509, and two salt bridges with HIS-345 and LYS-187 (Figure 2B). The ∆G of stabilization was found to be -1.16 kcal/mol, Kd of 0.141 M-1. Figure 2C showed that ginsenoside Rb1 was comfortably fitted at the active site cavity of ACE2. It interacted with SER-124, TYR-199, TRP-203, ASN-508, ASP-509 and TYR-510 through hydrogen bonds, while TYR-202, ASN-508 and TYR-510 were involved in hydrophobic interactions. The complex was stabilized by a ∆G of -4.72 kcal/mol, which corresponded to a Kd of 3.45 × 104 M-1. Ginsenoside F1 complex was stabilized by three hydrogen bonds with ASN51, and seven hydrophobic interactions with TYR-127, VAL-343, HIS-345 and PHE-504. The ∆G and Kd of ginsenoside F1 towards ACE2 were estimated to be -1.84 kcal/mol and 4.49 × 102 M-1, respectively. Based on the data, the presence of more hydrogen bonds in ginsenoside Rb1 seems the key factor for their high activity.