key: cord-0922976-jcgqa5v6
authors: Yu, Jiu-wang; Wang, Lu; Bao, Li-dao
title: Exploring the Active Compounds of Traditional Mongolian Medicine in Intervention of Novel Coronavirus (COVID-19) Based on Molecular Docking Method
date: 2020-05-15
journal: J Funct Foods
DOI: 10.1016/j.jff.2020.104016
sha: 9f577bf9d30e119ba748f339b36df1e48810912f
doc_id: 922976
cord_uid: jcgqa5v6

OBJECTIVE: This article intends to use molecular docking technology to find potential inhibitors that can respond to COVID-19 from active compounds in Mongolian medicine. METHODS: Mongolian medicine with anti-inflammatory and antiviral effects is selected from Mongolian medicine prescription preparations. TCMSP, ETCM database and document mining methods were used to collect active compounds. Swiss TargetPrediction and SuperPred server were used to find targets of compounds with smiles number. Drugbank and Genecard database were used to collect antiviral drug targets. Then the above targets were compared and analyzed to screen out antiviral targets of Mongolia medicine. Metascape database platform was used to enrich and analyze the GO (Gene ontology) annotation and KEGG pathway of the targets. In view of the high homology of gene sequences between SARS-CoV-2 S-protein RBD domain and SARS virus, as well as their similarities in pathogenesis and clinical manifestations, we established SARS-CoV-2 S-protein model using Swiss-Model. The ZDOCK protein docking software was applied to dock the S-protein with the human angiotensin ACE2 protein to find out the key amino acids of the binding site. Taking ACE2 as the receptor, the molecular docking between the active ingredients and the target protein was studied by AutoDock molecular docking software. The interaction between ligand and receptor is applied to provide a choice for screening anti-COVID-19 drugs. RESULTS: A total of 253 active components were predicted. Metascape analysis showed that key candidate targets were significantly enriched in multiple pathways related to different toxins. These key candidate targets were mainly derived from phillyrin and chlorogenic acid. Through the protein docking between S-protein and ACE2, it is found that Glu329/Gln325 and Gln42/Asp38 in ACE2 play an important role in the binding process of the two. The results of molecular docking virtual calculation showed that phillyrin and chlorogenic acid could stably combine with Gln325 and Gln42/Asp38 in ACE2, respectively, which hindered the combination between S- protein and ACE2. CONCLUSION: Phillyrin and chlorogenic acid can effectively prevent the combination of SARS-CoV-2 S-protein and ACE2 at the molecular level. Phillyrin and chlorogenic acid can be used as potential inhibitors of COVID-19 for further research and development.

Since December 2019, the epidemic situation of novel Coronavirus (COVID- 19) infection in China has developed rapidly. Up to now, the number of confirmed infections has exceeded 60,000 and spread to more than 20 countries and regions around the world. On January 31, 2020, the World Health Organization declared the COVID-19 epidemic as a public health emergency of international concern (PHIC) [1, 2] . However, at present, the research and development of virus vaccine is greatly lagging behind, and there is an extreme lack of effective therapeutic drugs against virus clinically. The world urgently needs to find and develop new therapeutic methods and drugs. Mongolian medicine has played a unique role in the prevention and treatment of new outbreaks of infectious diseases, especially in the prevention and treatment of SARS, H1N1, H7N9 and other epidemic situations, and has achieved very good clinical results. Mongolian medicine calls acute infectious diseases pestilence. Prevention and treatment of pestilence is an important part of Mongolian medicine. COVID-19 belongs to the category of "epidemic fever" and "sticky epidemic" in Mongolian medicine, and is a "pulmonary epidemic fever" caused by virus infection [3] . Against the spread of SARS-CoV-2, many Mongolian medical experts have proposed different Mongolian medicine prevention and treatment programs [4] . In order to provide Mongolian medicine plans with certain clinical basis and objective evidence in the first time, in view of the high homology of gene sequences between SARS-CoV-2 and SARS virus, and the great similarity of their onset characteristics, clinical manifestations and potential therapeutic targets, we intends to use the network pharmacology and molecular docking technology to screen compounds with clear anti-COVID-19 based on the experience and theory of Mongolian medicine in preventing and controlling major epidemics. Network pharmacology is a research strategy for multi-target and multi-channel interactions of drugs. Starting from the integrity and systematicness of interactions between drug targets and diseases, it uses computer methods to model multi-target activities on the basis of multi-level networks of diseases, genes and drugs. At the same time, it studies the biological basis of drugs acting on the body, which is a powerful tool for Mongolian medicine modernization research [5] . Molecular docking is a computational tool for predicting the binding ability and binding mode of proteins and ligands. Its principle is based on the "lock key model" of the interaction between proteins and small ligands, calculating and predicting the conformation and orientation of ligands at protein active sites, so as to judge the binding degree and play an important role in the target prediction of drug organisms [6] .There are many links and factors that affect the infection in SARS-CoV-2. According to previous studies, the infection routes of SARS-CoV-2 and SARS-CoV are the combination of S-protein of the virus and angiotensin converting enzyme (ACE2) in human body, which leads to the invasion of the virus into the body and causes disease [7] . In this study, molecular docking screening was carried out on ACE2, a key target protein in the process of virus infection, to obtain active compounds against coronavirus, providing reference for effective drug screening and new drug development.

This study mainly collected classic antipyretic and antiviral Mongolian medicine prescriptions, and used the involved Mongolian medicine to build a candidate Mongolian medicine database. With the help of the TCMSP database (http://tcmspw.com/), the chemical constituents of candidate Mongolian medicines were retrieved, and animal and mineral medicines such as cicada and gypsum were removed. Using TCMSP database, the pharmacokinetic (absorption, distribution, metabolism, exclusion, ADME) properties of main compound components were evaluated, and chemical components satisfying Oral Bioavailability (OB) ≥30% and Drug-Likeness property (DL)≥0. 18 were selected as candidate active components [8] . OB is directly related to bioavailability. DL refers to the similarity between the molecule to be tested and the drug molecule, i.e. the possibility of becoming a drug. The molecular structure of each active compound was confirmed by literature mining and PubChem (https://pubchem.ncbi.nlm.nih.gov/). domain has high homology with SARS-CoV, we downloaded the sequence file (FASTA format) (PDB:6ACD) of SARS-COV S-protein using RCSD protein database(www.rcsb.org), replacing Y with L at 442, F with L at 472, C with N at 479, and N with T at 487. The newly obtained SARS-CoV-2 sequence file is imported into Swiss-Model (https://swissmodel.expasy.org/), and thus the structural model of SARS-CoV-2 S-protein is constructed.

Docking of S-protein with ACE2 protein ZDOCK Server (http://zdock.umassmed.edu/) was used to rigidly dock SARS-CoV-2 S-protein with ACE2. The crystal structure of ACE2 was obtained from RCSD protein database (PDB: 2AJF). According to molecular composition and literature data, the main parameters of ZDOCK are shown in table 1. After the ligand-receptor complex model is obtained, the ZDOCK results are preliminarily screened using an empirical scoring function to obtain morphological near-natural conformational clusters of the receptor-ligand complex. The general formula of the scoring function is: SPSC+DE+ELEC = RELRPSC+ELEC LPSC+ELEC]+0.5XLM [RDE LDE], i.e. the scoring takes into account the factors of paired surface fit (PSC), desolvation reaction (DE) and electric potential (ELEC). Finally, PDBePISA database (https://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) was used to optimize and rearrange the conformational clusters, and the model with root mean square deviation (rootmeansquaredeviati0n.RMSD) less than 4A°was selected as the near natural conformation. The conformation was observed by pyMOL visualization software to obtain the key amino acid residues of the binding site. [15] . Autodock molecular docking software (version 2.5) is used to dock the active ingredients of traditional Chinese medicine with receptor protein to screen the effective ingredients against coronavirus. The detailed flow chart of this article is shown in fig. 1 .

The flow diagram for screening and verifying Mongolian medicine compounds.

In this study, based on the systematic review and analysis of Mongolian medicine prescriptions during the SARS period, the classic Mongolian medicine prescriptions were analyzed. A total of 13 Mongolian medicine preparations were collected, including 41 Mongolian medicines. By examining the compatibility of prescriptions, 11 Mongolian medicines with clear anti-inflammatory and antiviral effects were included in the analysis. Table 2 is the basic information of some active compounds. zanamivir, paramivir, lopinavir, ritonavir, amantadine, rimantadine, chloroquine, remdesivir and favipiravir ten drugs. 1047 known antiviral targets have been selected through the Drugbank database and Genecard database, and an antiviral target database has been constructed. Specific data was shown in schedule 4.

By comparing and analyzing the action targets of the above-mentioned collected active ingredients of traditional Chinese medicine with those related to antiviral drugs, 202 action targets of active ingredients of traditional Mongolia medicine with definite antiviral effects are summarized. The Metascape platform was used to perform GO annotation analysis and KEGG pathway analysis on the potential targets for antivirus. The threshold value P ＜ 0.01 was set to screen out the front GO annotation results and KEGG pathway. The results are shown in Fig2-B and the specific values are shown in table 3. The results showed that there were 20 signal pathways with high coincidence, and KEGG pathway annotation related to anti-toxic substances was selected to map the genes corresponding to the regulatory target protein directly to the pathways. The GO annotation has a large amount of data, among which, the top ones in the biological process are response to toxic substance, response to xenobiotic stimulus, Cellular Response to Nitrogen Compound. Through the enrichment analysis of KEGG pathway in Metascape database, two pathways closely related to immunity and anti-allotoxin are mainly IL-17 signaling pathway and PID ATF2 pathway. Kappa Score is an indicator of the extent to which two raters who are examining the same set of categorical data, agree and takes into account agreement occurring by chance. The nodes are connected to form a network by the similarity between terms (Kappa>0.3), and each node represents an enrichment term. The color of the node in Fig2-C indicates the cluster to which the node belongs. It can be seen that the terms belonging to the same cluster are closer and more closely related to each other. The color of the nodes in Fig2-D indicates the degree of enrichment (P value), and it can be seen that the more the number of genes is included, the more significant the P value is. 

Through the Metascape platform, all genes are connected to the whole protein interaction network (Fig3-B) . Seven different colors represent the module substructures identified in the interconnection network, and the formed modules are abstracted from the full-connection interconnection network to form Fig3-C. The protein functions of specific modules are shown in Table 4 . Key genes corresponding to regulatory target proteins are directly mapped onto pathways, and pathways enriched by drug targets are considered as pathways for drug regulation. It can be found that in MCODE 1 and MCODE 5, the protein function annotation is the cell's response to toxin and heterologous biological stimulation, and 36 key targets involved mainly come from two compounds, Phillyrin and chlorogenic acid. 

After SARS-CoV-2 S-protein RBD domain docked with ACE2 via ZDOCK, 8 receptorligand complex models were obtained. Energy optimization and model screening were carried out for all conformations using PDBePISA database, and the specific data are shown in Table 5 . The best model is the one with the largest surface area, the smallest surface energy and the most hydrogen bonds [16] . After comprehensive consideration, complex2 was selected as the near-natural model (Fig 5-B) . Analysis of the key binding sites of SARS-CoV-2 and ACE2 showed that SARS-CoV-2 S-protein Arg426 and Tyr436, respectively, combined with Gln325/Glu329 and Asp38/Gln42 of ACE2 through hydrogen bonds and formed an interaction interface with adjacent residues (Fig  6) , which is consistent with previous research results of Hao Pei team [17] . 

In this study, two key active compounds phillyrin and chlorogenic acid screened by network pharmacology were verified. The 3D structures were imported into AutoDock and docked with ACE2. Their interactions with surrounding key residues and their binding at the active site are shown in Fig7, and the energy values of the compounds shown in the docking results are shown in Table 6 . Molecular docking results showed that phillyrin and Gln325 of ACE2 were mainly combined in the form of hydrogen bonds and had good binding activity, which indicated that phillyrin could hinder the binding of SARS-CoV-2 S-protein RBD domain and ACE2 at Gln325/Glu329 , and the docking energy value was smaller and the binding was more stable. Chlorogenic acid combines with ACE2 Gln42/Asp38 in the form of hydrogen bonds. Compared with SARS-CoV-2 and ACE2 binding model, the docking energy is smaller and the binding is more stable, which indicates that chlorogenic acid can hinder the binding of SARS-CoV-2 S-protein RBD domain and ACE2 at Gln42/Asp38. The above results show that the combination of phillyrin and chlorogenic acid will probably hinder the binding of ACE2 and S-protein more effectively. 

Since the end of December 2019, COVID-19 has been ravaging the whole country. Currently, there is still a lack of specific drugs for the treatment of COVID-19, and the existing chemical drugs can only alleviate some symptoms. The development and research of new drugs based on clinical experiments are long and expensive, which is difficult to apply to clinical treatment in time. Moreover, SARS-CoV-2 has high infectivity and variability. The research requires high-level laboratory conditions, which greatly limits drug screening. Therefore, computer-aided drug design has fast speed, save drug development costs, and is suitable for large-scale screening of chemical components of traditional Mongolia medicine. Molecular docking can simulate the force between the three-dimensional structure of the receptor and the ligand to find a low-energy binding mode between the ligand and the active site of the receptor, which is fast, efficient and low in cost. Forsythia is the dried fruit of Forsythia suspense (Thunb.) Vahl of the osmanthaceae family. It has the functions of clearing away heat and detoxification, reducing swelling and loosening，and is commonly used for treating carbuncle, scrofula, breast abscess, etc [18] . Phillyrin is an active ingredient in Forsythia and a quality control ingredient in Forsythia and its preparations. Its pharmacological effects are extensive, such as clearing away heat and toxic materials, antivirus, antioxidant, antibacterial and antiinflammatory effects [19, 20, 21] . RT-PCR showed that phillyrin can reduce the copy number of NP gene of influenza A virus. The Jian Sun team believed that phillyrin may inhibit NP gene replication by inhibiting the combination of influenza A virus NP and viral RNA to form NP complexes, and may also inhibit the transcription level or posttranslational modification of NP. This problem is helpful for further research [22] . Honeysuckle is the dried flower bud of Lonicera japonica Thunb. of Caprifoliaceae and other plants of the same genus, which has the effect of clearing away heat and toxic materials, and belongs to the precious traditional Mongolia medicine under the Strict management in China [23, 24] . In April 2018, Honeysuckle was explicitly listed in the "Medicine and food homologous food" catalogue by the Ministry of Health of the People's Republic of China. Chlorogenic acid is the main active ingredient of Mongolian medicine honeysuckle, which is an ester substance formed by caffeic acid and quinic acid. It is easy to isomerize through hydrolysis or ester group migration in the extraction process [25, 26] . Chlorogenic acid is very high in Eucommia ulmoides, honeysuckle, coffee bean, blueberry, apple and potato containing phenolic acids. As the main active ingredient of the Mongolian medicine honeysuckle, chlorogenic acid has been proven to have significant pharmacological effects, such as antioxidant, antitumor, hypoglycemic, anticoagulant, antiviral effects, etc [27, 28] . Studies by Wang Xuebing and others have shown that chlorogenic acid plays a significant role in preventing viral infection in the early stages of virus growth, and also has a strong inhibitory effect on porcine parvovirus, showing that chlorogenic acid has a strong antiviral effect [29] . [32] . It also has anti-inflammatory, antipyretic, analgesic, and anti-allergic effects. There are more than 400 Chinese and Mongolian medicine preparations involving honeysuckleforsythia drug pairs in the existing Chinese patent medicines on the market, such as Lianqiao baidu pill, Yinhua ganmao granule, Zhizi Jinhua pill, etc [33] . Meiyi Zhang et al studied the anti-H1N1 influenza virus effect of Jinqiao Tablets in vivo, and found that Jinqiao Tablets can significantly inhibit the increase of lung index in mice, reduce the viral load of lung tissue, and increase the level of γ-IFN, thus having obvious therapeutic effect [34] . Pan Qu et al. conducted in vitro studies on the anti-influenza virus effects of honeysuckle, forsythia, burdock and their extracts, which are the main drugs of Yinqiao Powder, and found that the single drugs and their compatible extracts have good anti-influenza A virus proliferation effects, and are correlated with drug concentration and duration of action [35] . Studies on the effect of honeysuckle-forsythia on respiratory syncytial virus show that Yinqiao powder has the effect of inhibiting respiratory syncytial virus, and the protective effect of drug-containing serum combined with the two drugs on cells is better than that of drug-containing serum of two single drugs [36] . Yinqiao Powder can reduce the lung index of mice [37] , reduce the damage of nasal mucosa epithelial cells and lung tissue. Its improvement effect on respiratory system of RSV infected mice is comparable to that of positive control drug ribavirin. In addition, in vitro cell experiments verified the improvement effect of Yinqiao Powder on RSV infection, and its mechanism of action is related to the activation of NALP3

inflammasome. An anti-RSV virus study conducted on Yinhua Ganmao Granules found that the half-toxic concentration of this preparation is 2.3937 mg / mL, the halftherapeutic concentration is 52.03 μg / mL, and the therapeutic index is 46.01, indicating that Yinhua Ganmao Granules have better inhibitory effect on RSV [38] . In the study of antipyretic and anti-inflammatory effects of honeysuckle, forsythia single medicine and compatibility, Jiarui Wu et al studied the mechanism of action of honeysuckle-forsythia drug pair by network pharmacology [39] . The diseases involved are mainly inflammatory diseases, which showed that the compatibility of the two drugs is consistent with the efficacy of clearing away heat and toxic materials. Although water decoction alone has certain anti-inflammatory effect, it has no statistical difference with the model group, and the anti-inflammatory effect is strongest when the two are used in combination at a ratio of 1:1 [40] . For antipyretic effect, the research results showed that forsythia alone has no antipyretic effect, and the antipyretic effect of honeysuckle and forsythia 1:1 compatibility is the most obvious, the combination of the two can reduce the level of IL-6 in experimental rats and play a certain antipyretic effect [41] .

Animal experiments have verified that in the antipyretic and anti-inflammatory effects, the combination of honeysuckle and forsythia can play a synergistic role.

To sum up, the active compounds phillyrin and chlorogenic acid have been explored to block the binding of SARS-CoV-2 S-protein and ACE2 by network pharmacology and molecular docking methods. The aim is to provide reference for further development of Mongolian medicine's treatment plan for COVID-19 epidemic. However, due to the lack of consideration of the content of chemical components, insufficient understanding of viruses and diseases, and limitations of molecular docking itself, the obtained results may have deviations. Later research should also be verified at multiple levels through pharmacodynamic evaluation, metabonomics, single target, etc.

In this study, a research strategy combining network pharmacological analysis, protein docking and molecular docking virtual computation was adopted. It was found that phillyrin and chlorogenic acid could block the combination of SARS-CoV-2 S-protein and ACE2 at the molecular level. Both can be used as potential inhibitors of SARS-CoV-2 for further research and development. The relevant research results of this experiment will provide theoretical basis for phillyrin and chlorogenic acid to resist SARS-CoV-2, and also provide methodological reference for the mechanism research of antiviral active ingredients of traditional Mongolia medicine.

Funding

COVID-19)

Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study

Diagnosis and Treatment Plan for Pneumonia Infected in COVID-19 (Trial Fourth Edition

Observation on Clinical Effect of Lianhua Qingwen Capsule in Treating Influenza

Study on the material basis, target and characteristics of Rongjin Niantong Decoction in the treatment of Sciatica by computer simulation

Revealing synergistic mechanism of multiple components in compound Fengshiding capsule for rheumatoid arthritis therapeutics by network pharmacology

Structure of SARS coronavirus spike receptor-binding domain complexed with receptor

Feature selection and syndrome prediction for liver cirrhosis in traditional Chinese medicine

Delivering QOS with security of encrypted tracer and checker model in MANET

SwissTargetPrediction:a web server for target prediction of bioactive small molecules

SuperPred: drug classification and target prediction

Mystery virus found in Wuhan resembles bat viruses but not SARS, Chinese scientist says

Dipeptidyl peptidase 4 is a functional receptor for the emerginghuman coronavirus-EMC

SWISSMODEL: an automated protein homology-modeling server

RajashankarKR ， eta1 ． Crystalstructureof mousecoronavim sreceptor-binding domain complexed with its murine receptor

Evolution of the COVID-19 from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission

Discussion on the drug pair with forsythia as the core and its compatibility rule in the ancient prescription of warm disease study

Forsythin inhibits lipopolysaccharide-induced inflammation by suppressing JAKSTAT and p38 MAPK signalings and ROS production

Antioxidant and antibacterial activity of two compounds (forsythiaside and forsythin) isolated from Forsythia suspensa

Study on the Effect of Forsythin on Nucleoprotein Gene Expression of Influenza A Virus

Anti-herpes simplex virus effect of chlorogenic acid in vitro

Research Progress on Extraction and Application of Chlorogenic Acid

Study on antiviral effect of Senecio cannabifolius extract and its effective components

On Modern Clinical Application of Yinqiao Powder

Medication Rules of "Feire" Syndrome in Ancient Books of Epidemic Febrile Disease Based on Data Mining

Study on Anti-influenza Virus Effect of Jinqiao Tablets

Comparative Study on the Inhibitory Effect of Extracts from Yinqiao Powder on Influenza Virus in Vitro

Pharmacodynamics Study of Shuanghuanglian and Its Decomposed Prescription on Anti-Respiratory Syncytial Virus

Effect of Yinqiaosan on respiratory system model of respiratory syncytial virus infection in mice

Preparation of Yinqiao Kanggan Granules

Analysis of Action Mechanism of Honeysuckle-Forsythia Suspensa Based on Network Pharmacology

Study on the Anti-inflammatory and Antipyretic Effects of Honeysuckle, Forsythia Suspense and Yinqiao Drugs on

Experimental Study on the Antipyretic Mechanism of the Compatibility of Honeysuckle and Forsythia

A research strategy combining network pharmacological analysis, protein docking and molecular docking virtual computation was adopted

Through the protein docking between S-protein and ACE2, it is found that

It was found that phillyrin and chlorogenic acid could block the combination of SARS-CoV-2 S-protein and ACE2 at the molecular level. Both can be used as potential inhibitors of SARS-CoV-2 for further research and development

Not applicable.

Ethics approval and consent to participate Not applicable.

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

The manuscript is approved by all authors for publication.

The authors declare no competing financial interests.

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. We confirm that the mentioned received grants in the "Acknowledgement" section, did not lead to any conflicts of interest regarding the publication of this manuscript.

Title of the article: Exploring the Active Compounds of Traditional Mongolian Medicine in Intervention of Novel Coronavirus (COVID-19) Based on Molecular Docking Method. On behalf of, and having obtained permission from all the authors, I declare that: a．Jiuwang Yu designed and analysed the data and wrote the manuscript; b．Lidao Bao provided technical assistance and advised on study design and data interpretation; c．Lu Wang provided intellectual input and Supervised the overall study. All authors read and approved the final manuscript.

Title of the article: Exploring the Active Compounds of Traditional Mongolian Medicine in Intervention of Novel Coronavirus (COVID-19) Based on Molecular Docking Method. On behalf of, and having obtained permission from all the authors, I declare that: a. the material has not been published in whole or in part elsewhere; b. the paper is not currently being considered for publication elsewhere; c. all authors have been personally and actively involved in substantive work leading to the report, and will hold themselves jointly and individually responsible for its content; d. our research did not include any human subjects and animal experiments, the relevant ethical safeguards do not apply to this study. I testify to the accuracy of the above on behalf of all the authors.Lidao Bao 29-Feb-2020