Anti–coronavirus disease 2019 (COVID‐19) targets and mechanisms of puerarin

Abstract The present study aimed to uncover the pharmacological function and underlying mechanism of puerarin as a potential treatment for COVID‐19, using an in silico methodology, including network pharmacology and molecular docking. The pivotal targets of puerarin to treat COVID‐19 were identified and included the epidermal growth factor receptor (EGFR), tumour necrosis factor (TNF), tumour protein p53 (TP53), caspase 3 (CASP3), RELA proto‐oncogene (RELA), Fos proto‐oncogene (FOS), caspase 8 (CASP8), prostaglandin‐endoperoxide synthase 2 (PTGS2), interleukin 2 (IL2), protein kinase CB (PRKCB), B cell lymphoma/leukaemia gene‐2 (BCL2), protein kinase CA (PRKCA), nitric oxide synthase 3 (NOS3) and peroxisome proliferator–activated receptor gamma (PPARG). Functionally, the anti–COVID‐19 action of puerarin was associated with the suppression of oxidative stress and inflammatory cascades, and cell apoptosis. The signalling pathways of puerarin to treat COVID‐19 included modulation of the pathways of apoptosis, IL‐17 signalling, mitogen‐activated protein kinase (MAPK) signalling and TNF signalling. Molecular docking data illustrated the binding capacity of puerarin with COVID‐19 and the effective anti–COVID‐19 activity of puerarin. Taken together, our current network pharmacology–based findings revealed the pharmacological role of puerarin in the treatment of COVID‐19. Furthermore, the bioinformatic findings elucidated that some of these pivotal targets might serve as potential molecular markers for detecting COVID‐19.


| INTRODUC TI ON
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogenic agent responsible for causing COVID-19, a severe and life-threatening epidemic disease that has led to a massive economic burden and increasing death toll in the world. 1 As an evolving epidemic, COVID-19 is rapidly growing and spreading in several countries, including the United States, Brazil and India. 2 Epidemiological evidence suggests that the trend of COVID-19 infection is difficult to control because of insufficient management against COVID-19, characterized by huge rates of cases in different countries. 3 In the United States, COVID-19 has already affected millions of people and is characterized by an increasing incidence and death rate. 4 Pathologically, the main clinical characteristics of COVID-19 are related to inflammation, cytokine storms, immunodeficiency and infection. 5 In clinical management against COVID-19, the common therapy may involve supportive care, 6 such as breathing assistance, as the development of an effective drug and new vaccine is still ongoing. 7 Traditionally, Pueraria montana (Gegen) is found to relieve muscle fever, neutralize alcoholism-related hypoglycaemic and hypolipemic actions. 8 Puerarin, rich in Pueraria montana, exerts evident pharmacological benefits, such as preventing coronary heart disease and anti-alcoholism and anti-diabetic effects. 9 In our previous preclinical studies, puerarin was found to have potent hepatoprotection, neuroprotection and anti-dysmetabolism effects. [10][11][12][13] It has been reported that puerarin may function as a natural amoebicidal agent to manage Acanthamoeba-induced pneumonia. 14 In addition, puerarin plays an important role in anti-inflammatory action, including prevention of acute lung injury by reducing inflammatory stress. 15,16 However, there is currently no specific drug for the treatment of COVID-19. Although some antiviral drugs and traditional Chinese medicines have been used in the clinical treatment of COVID-19, its therapeutic effectiveness is not clear. Collectively, we aimed to determine and identify the curative effect of puerarin in the treatment of COVID-19 and the therapeutic mechanisms involved. It is of great interest to use bioinformatic and computational methods based on network pharmacology to unveil the potential agent to treat illness. 17 Using the network pharmacology strategy, we have reported the pharmacological targets and molecular pathways of bioactive agents against complex diseases. 18,19 Thus, in this bioinformatic report, we intended to reveal the component-target-pathway network and pharmacological mechanism of puerarin in the treatment of COVID-19.

| Screening of the target of puerarin in the treatment of COVID-19
The total established target genes of puerarin were selected using the effective tools of traditional Chinese medicine systems pharmacology (TCMSP), Swiss Target Prediction and SuperPred.
Meanwhile, other COVID-19-nosopoietic genes were acquired using the gene-disease associations (DisGeNET) and GeneCards databases. Additionally, these acknowledged genes of puerarin and COVID-19 were mapped prior to emendation using the UniProt tool.
After functional enrichment analysis using FunRich software, all anti-COVID-19 biotargets of puerarin were screened and identified, as reported elsewhere. 20,21

| Screening of pivotal targets of puerarin to treat COVID-19 and construction of PPI network
The total acknowledged targets of puerarin and COVID-19 were used to further determine and construct a functional protein association network via the STRING database following a specific algorithm. In addition, these merged targets of puerarin and COVID-19 were used to construct a network of protein-protein interactions (PPI) using the Cytoscape software. As revealed by topological parameters using the Network Analyzer tool, the pivotal targets of puerarin to treat COVID-19 were identified and visualized. 22,23

| Enrichment analyses in biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of intersection targets
Using the R language packages, such as 'ClusterProfiler', 'ReactomePA', 'org.Hs.eg.Db' and 'GOplot' in the R language

| Molecular docking analysis
Using the chemical-protein binding method, the pivotal targets were screened out and identified for puerarin-based molecular docking analysis. After searching for specific proteins through the PDB database, 5R84 protein was selected to dock with the puerarin compound.

| Findings of putative genes of puerarin and COVID-19
As revealed in Figure 1, 393 COVID-19-morbid genes were selected and detected. A total of 138 putative genes of puerarin were specified following the databases. As shown in the Venn diagram, 34 shared genes of puerarin to treat COVID-19 were screened and identified. Furthermore, these mapped genes were reused to plot an interaction network.

| Identification of all pivotal targets
All the mapped intersection proteins were input into Cytoscape 3.

| Enrichment findings of pivotal targets
Following the assays using R language-related packages, the anti-COVID-19 molecular pathways from puerarin-achieved pivotal targets F I G U R E 1 All common genes of puerarin and COVID-19 were identified in a Venn map prior to further identification of the shared targets of puerarin to treat COVID-19. Total 34 shared targets of puerarin to treat COVID-19 were harvested and plotted for construction of a PPI network  Figure 4A,B, Table S3).

| Construction of network diagram
Further, the network visualization of puerarin-target-biological process (BP)-KEGG-COVID-19 was created using Cytoscape 3.7.1 software, as shown in Figure 5.

F I G U R E 5
In details, an integrated network map from pivotal targets was plotted and revealed the intersection association of targetdisease-function-pathway in puerarin to treat COVID-19

| D ISCUSS I ON
In our current study, the in silico findings from the network phar- also related to apoptotic cell death. 28 Prostaglandin-endoperoxide F I G U R E 6 Molecular docking findings suggested that the binding capacity of puerarin against COVID-19 was significant in the pivotal target/protein. The biostructural original ligands binding to targeting protein with effective docking energy, as shown in A and B synthase (PTGS) is responsible for the prostanoid biosynthesis involved in inflammation and mitogenesis when it is induced by specific stimulatory genes. 29 Some interesting data indicate that targeting suppression of PGE2 synthesis is likely to reduce bacterial infection in mice. 30 Protein kinase C (PKC), including PRKCB and PRKCA, is involved in diverse cellular signalling pathways, including those involved in cancer. 31 The proteins encoded by PRKCB and PRKCA genes have been reported to be associated with numerous cellular functions, such as B cell activation, apoptosis induction and endothelial cell proliferation. 32 NOS3 (nitric oxide synthase 3) may induce nitric oxide release for the regulation of angiogenesis and cell proliferation. 33 Interestingly, NOS3 may functionally suppress sepsis-caused systemic inflammation and myocardial dysfunction in mice. 34 Collectively, there is no associated investigation for the associations between FOS, PTGS, PRKCB, PRKCA, NOS3 and COVID-19.
Therefore, the current in silico findings using network pharmacology uncovered all pharmacological targets, functions and signalling pathways of puerarin to treat COVID-19, marked by inhibition of cytonecrosis-and inflammation-associated signalling pathways, such as the IL-17 signalling pathway, TNF signalling pathway, MAPK signalling pathway and HIF-1 signalling pathway. Potentially, puerarin may be a promising active compound against COVID-19. However, it needs to be confirmed by future experimental works.

| CON CLUS ION
In silico approaches based on network pharmacology and molecular docking were used to harvest the mapped and pivotal biotargets,

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article.