Discovery of a potent angiotensin converting enzyme 2 inhibitor from Chinese Medicinal and Edible Plant via Docking-based Virtual Screening


 COVID-19 has outbreaked in Wuhan city, Hubei province of China since December 30th 2019, and spread nationwide and widely spilled over to other countries around the world that has been declared a public health emergency. However, there is no specific drug for the treatment of the disease. Therefore, identifying effective antiviral drugs to combat the disease is urgently needed. Angiotensin converting enzyme 2 (ACE2) has become the promising target to discovery new antiviral drugs to treat COVID-19, we have attempted to discover novel ACE2 inhibitors through ligand-based virtual screening. Finally, eight compounds were selected and tested ACE2 kinase inhibitory assay using fluorescence assays method. The results showed that four compounds (monoammonium glycyrrhizinate, glycyrrhizic acid methyl ester, ginsenoside Rg6 and ginsenoside F1) from 101 kinds of Chinese medicinal and edible plants which could inhibit ACE2 activity in vitro. Further efforts on chemical modification of these lead compounds are undergoing can lead to discover better agents against COVID-19.


Introduction
The 2019 novel coronavirus (2019-nCoV), SARS-CoV-2, was rstly reported on December 30 th in Wuhan city, Hubei province of China [1]. The disease was recently named as COVID-19 by the World Health Organization (WHO). COVID-19 has been spread nationwide and widely spilled over to other countries around the world has posed a serious global public health emergency [2]. According to WHO on March 15 th 2020, there has been over outbreak of COVID-19 in 152 counties, leading to 173344 con rm cases and including 7019 deaths. However, there is no speci c drug against COVID-19. Therefore, developing effective antiviral agents to combat the disease is urgently needed.
Up to date, researchers reported that the Cryo-EM structure of COVID-19 and the crystal structure of COVID-19 spike receptor (S) binds angiotensin converting enzyme 2 (ACE2) complex [2][3][4][5]. Similar with syndrome-related coronavirus (SAR-CoV), S protein of COVID-19 contains the receptor binding domain (RBD) binds to the ACE2 ( Figure 1A-1B) [6]. The inhibitor of ACE2 is likely to change the conformation of ACE2 and block the interaction between S protein and ACE2 receptor. Thus, ACE2 has become the promising target to discovery new antiviral drugs to treat COVID-19, and some inhibitors targeting ACE2, such as MLN-4760, DX600, 4S-16659 and NAAE [7][8][9][10]. However, due to its undesirable side effects, such as renal failure, persistent cough and angioedema, attempts to use the synthetic ACE2 inhibitors in clinic have been mostly unsuccessful. Therefore, more efforts are made for safe and speci c drugs development.
Natural products from plant have served as important sources of lead compounds for developing drugs. Notably, many studies found that Traditional Chinese Medicine (TCM) extract effectively inhibited the activity of ACE [11,12]. Moreover, medicinal and edible plants are safe and effective play an important role in supporting healthcare both in China and around the world. In this study, we used docking screening to identify more natural ACE2 inhibitors from 101 kinds plants that are used in China medicine and as food supplements, therefore providing a potential prevention/treatment agents against COVID-19.

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 nd the best structures for protein have been used in many studies [15][16][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) [2] was most similarity with 1R4L (see Supplementary data Figure S1). The active site was de ned 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 a nity (K i ) were calculated between the bioactive and docking conformations ( Table 1). As shown in Table 1, RMSD values were less 0.3 nm and K i 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.) [19]. By applying a docking score cutoff of ≥ 9.0 kcal/mol, we identi ed 33 compounds (~5%) with the maximum scores ( Table 2). These compounds were used for further assessing the physiochemical.

ACE2 kinase Inhibitory Assay
To assess the inhibitory potency of the identi ed 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][27][28][29]. Ginsenoside Rg6 and ginsenoside F1 show antitumor, antioxidant and anti-in ammatory effects in vitro [30][31][32][33]. Previous study has reported that ginsenoside Rb1 exerted inhibitory activity against ACE [34]. 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-in ammatory, antiulcer, 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.

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 a nity (K d ) were estimated to be -1.63 kcal/mol and 6.23 × 10 2 M -1 , respectively. We found that glycyrrhizic acid methyl ester binds at the site of ACE2 by forming ve 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, K d of 0.141 M -1 . Figure 2C showed that ginsenoside Rb1 was comfortably tted 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 K d of 3.45 × hydrophobic interactions with TYR-127, VAL-343, HIS-345 and PHE-504. The ∆G and K d of ginsenoside F1 towards ACE2 were estimated to be -1.84 kcal/mol and 4.49 × 10 2 M -1 , respectively. Based on the data, the presence of more hydrogen bonds in ginsenoside Rb1 seems the key factor for their high activity.

In vitro ACE2 inhibitory activity assay
In brief, ACE2 acitivity assay was performed using 10 μM uorogenic peptide substrate Mca-R-P-PG-F-S-A-F-K (Dnp)-OH in balck 384-well opaque plates with 0.1% DMSO solution, 1µM captopril and 100 µM test compounds. 0.2 ng/μl recombinant human ACE2 protein was added to the reaction mixtures. The uorescence value was measured every other 2 minutes. The enzymatic activity was recorded on a SpectraMAX Gemini uorescence spectrophotometer at an excitation wavelength of 328 nm and an emission wavelength of 392 nm. Fluorescence was monitored at 36 s intervals for 15 min [7,17]. The percentage of inhibition was calculated as follows: where S com , S con and S DMSO were the slope value of the compound group, control group and DMSO group. The data represented the mean of three assay results.

Conclusion
In conclusion, we have discovered four compounds (monoammonium glycyrrhizinate, glycyrrhizic acid methyl ester, ginsenoside Rg6 and ginsenoside F1) from 101 kinds of Chinese medicinal and edible plants which could inhibit ACE2 activity in vitro. These compounds had strong potential to be further