The herb-property-flavor-meridian tropism network for CYM prescription
Based on TCM theory, the five herbs in CYM were classified according to their property, flavor and meridian tropism (Table 1 and Figure 2). A network was constructed among elements of the TCM classification system (Table 1) and the five herbs of the CYM prescription via R software. The connection degree of lung, heart, stomach and small intestine is the highest in the meridian tropism group, and the corresponding connection values are four, three, two and two, respectively. Four herbs (Houttuyniae Herba, Scutellariae Radix, Lonicerae Japonicae Flos, Forsythiae Fructus) in CYM are associated with the lung meridian. Among the flavor group, the greatest degree of connection is bitter, with three degrees of association.
Screening potential targets of CYM
To identify the active compounds of CYM, two classical ADME parameters, OB%≥30% and DL≥0.18, were used for screening. Through mining the TCMSP, a total of 594 potential compounds were screened from five traditional Chinese medicines (Supplemental Tabel 1). After that, we inputted the molecular name and SMILE number of the active ingredients into the Drugbank and STITCH databases to obtain 151 and 80 putative targets of CYM, respectively. The putative targets included PTGS1, IL–6, JUN, TNF, IFNG, IL–2 and so on. Detailed information about the potential targets of CYM was shown in (Figure 3A, Supplemental Table 2).
The acquisition of known therapeutic targets for COVID–19
Genes related to COVID–19 were retrieved from the DisGeNET database and MalaCards database using the keywords “SARS” and “MERS”. In the study, a total of 260 known therapeutic targets were collected from DisGeNET database and 144 known therapeutic targets were collected from MalaCards database. After eliminating redundant targets, a total of 351 known therapeutic targets were collected (Figure 3B). Then the potential targets in CYM was mapped to the COVID–19 targets to obtain 32 therapeutic genes using the ImageGP platform, and a Venn diagram was drawn (Figure 3C and Table 2).
PPI networks construction and core genes screening
PPI network was used to explore the function of diverse targets in CYM and COVID–19. A acquisition of 32 targets were inputted into the STRING database, the medium confidence score was set as 0.400 for further analysis, and then visualized by Cytoscape 3.7.1. There were 32 protein nodes and a total of 225 interactive connecting lines in the PPI network. The average node degree of freedom is 14.1, and the avg. local clustering coefficient is 0.758. Among all core targets, the bigger the deeper, the more important it is. Further analysis from cytoHubba revealed that the degree of tumor necrosis factor (TNF), interleukin 6 (IL–6) and interleukin 1β (IL–1β) is 30, 28 and 25 respectively, which were closely related to the inflammatory response (Figure 4).
Drugs-Compounds-Targets network analysis
To elucidate the potential mechanism of CYM in the treatment of COVID–19, we used Cytoscape 3.7.1 software to build the Drugs-Active compounds-Targets network. The hexagon nodes represent five traditional Chinese medicines, the ellipse nodes represent 103 active ingredients and the diamond nodes represent 32 overlapping targets for disease. The edges indicate that nodes can interact with each other. Further analysis of the network topology shows that the centralization and heterogeneity are 0.339 and 1.602 respectively, which indicates that the compound-target space has a tendency for certain compounds and targets. Therefore, the network contains some core ingredients with multi-targets, such as Quercetin (degree = 75), Luteolin (degree = 24), Beta-sitosterol (degree = 28), Wogonin (degree = 18), Acacetin (degree = 10), Kaempferol (degree = 24), which may play a crucial role in the treatment of COVID–19 (Figure 5).
Gene ontology enrichment analysis
To verify whether the 32 target genes are related to COVID–19, we entered the candidate targets into the STRING platform for GO functional enrichment and annotation. In the study, a total of 750 biological processes, 52 molecular functions, and 25 cellular components were obtained. The top 10 entries were selected based on false discovery rate (FDR) <0.05 and the number of enriched genes, and then visualized by Cytoscape (Figure 6). The X-axis indicates the number of enriched genes for the term, and the Y-axis indicates the GO term. The results demonstrated that the biological processes mainly involved response to stimulus (32 targets), cellular process (32 targets), biological regulation (30 targets) (Figure 6A, B); the molecular functions mainly involved Binding (31 targets), Protein binding (26 targets), Catalytic activity (17 targets) (Figure 6C, D); the cellular components mainly involved Intracellular (30 targets), Extracellular region (24 targets), Extracellular space (20 targets) (Figure 6E, F). The GO enrichment analysis results showed that the active compounds of CYM could participate in various cellular processes, so as to treat COVID–19.
The KEGG pathway enrichment analysis
The selected targets were inputted into KOBAS database to carry out pathway enrichment analysis. A total of 183 signaling pathways were obtained, and the top of 20 entries were screened according to the number of enriched targets (Figure 7A, B). The signaling pathways were listed as follows: Pathways in cancer, Inflammatory bowel disease (IBD), IL–17 signaling pathway, Chagas disease (American trypanosomiasis), AGE-RAGE signaling pathway in diabetic complications, C-type lectin receptor signaling pathway, Yersinia infection, Fluid shear stress and atherosclerosis, Tuberculosis, Cytokine-cytokine receptor interaction, Leishmaniasis, Th1 and Th2 cell differentiation, T cell receptor signaling pathway, Th17 cell differentiation, Jak-STAT signaling pathway, NOD-like receptor signaling pathway, Human cytomegalovirus infection, Pertussis, Toll-like receptor signaling pathway, TNF signaling pathway. These results suggest that the effective pharmacological ingredients in CYM may treat COVID–19 through alleviating inflammatory responses and enhancing immune pathways. The inflammatory bowel disease (IBD) and IL–17 signaling pathway, which play a crucial role in treating disease were annotated (Figure 7C, D).
Analysis of molecular docking and binding modes results
It is worth noting that we have made several milestone discoveries, including the crystal structure of 3CLpro, which can serve as candidate molecular target for SARS therapy(12) . To evaluate the targeting of several active ingredients in CYM to COVID–19 3CLpro, we selected the top six ingredients with larger degrees from the network diagram for molecular docking and binding energy analysis (Table 3 and Figure 8). The binding energy of the core active ingredients of CYM are all less than –5 kJ/mol, of which the lowest binding energy of active compounds with COVID–19 3CLpro is beta-sitosterol (–8.63 kJ/mol).