SARS-CoV-2 intervened by NSAIDs: A network pharmacology approach to decipher signaling pathway and interactive genes

Background: Non-Steroidal Anti-Inammatory Drugs (NSAIDs) showed promising clinical ecacy toward COVID-19 patients as painkillers and anti-inammatory agents. However, the prospective anti-COVID-19 mechanisms of NSAIDs are not evidently exposed. Therefore, we intended to decipher the most potent NSAIDs candidate(s) and its novel mechanism(s) against COVID-19 by network pharmacology. Method: FDA (U.S. Food & Drug Administration) approved twenty NSAIDs were used for this study. Genes related to selected NSAIDs and COVID-19 related genes were identied by the Similarity Ensemble Approach, Swiss Target Prediction, and PubChem databases. Venn diagram identied overlapping genes between NSAIDs and COVID-19 related genes. The interactive networking between NSAIDs and overlapping genes was analyzed by STRING. RStudio plotted the bubble chart of KEGG pathway enrichment analysis of overlapping genes. Finally, the binding anity of NSAIDs against target genes was determined through molecular docking analysis. Results: Geneset enrichment analysis exhibited 26 signaling pathways against COVID-19. Inhibition of proinammatory stimuli of tissues and/or cells by inactivating RAS signaling pathway was identied as the key anti-COVID-19 mechanism of NSAIDs. Besides, MAPK8, MAPK10, and BAD genes were explored as the associated genes of the RAS. Among twenty NSAIDs, 6MNA, rofecoxib, and indomethacin revealed promising binding anity with the highest docking score against three identied genes, respectively. Conclusions: Overall, our proposed three NSAIDs (6MNA, rofecoxib, and indomethacin) might block the RAS by inactivating its associated genes, thus may alleviate excessive inammation induced by SARS-CoV-2. NSAIDs-genes networking analysis genes. The results of KEGG genes that 26 signaling pathways were associated with the and of the COVID-19 symptoms. The correlations of 26 signaling pathways with COVID-19 symptoms were succinctly discussed as follows. PPAR (Peroxisome Proliferator-Activated Receptor) signaling pathway: A report shows that PPARγ (Peroxisome Proliferator-Activated Receptor-gamma), PPARα (Peroxisome Proliferator-Activated Receptor-alpha), and PPARβ/δ (Peroxisome Proliferator-Activated Receptor-beta/delta) agonists have anti-inammatory and immunomodulatory functions 16 . MAPK (Mitogen-Activated Protein Kinase) signaling pathway: The mechanisms of p38 MAPK inactivation might be a signicant therapy against the SARS infected cells 17 . Additionally, MAPK stimulates cytokine production such as IL-10 (Interleukin 10), TNF- α (Tumor Necrosis Factor-Alpha), IL-4 (Interleukin 4), and IFN- γ (Interferon gamma) 18 . This report shows a coincidence with our suggested strategy in this study. ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) signaling pathway: ErbB signaling reduces the proinammatory activation in cardiac cells 19 . RAS (Renin Angiotensin System) signaling pathway: Inactivation of RAS can reduce tissue damage in COVID-19 patients. In addition, ACE (Angiotensin Converting Enzyme) antagonists block the response of RAS system 20 . cGMP-PKG (Cyclic GMP-Protein Kinase G) signaling pathway: The activation of cGMP-PKG signaling inhibits inammatory response in the prostate, and also decreases CCL5 (C-C Motif Chemokine Ligand 5) release in CD8 + T cells (Cluster of Differentiation 8 T cells) 21 . cAMP (Cyclic Adenosine Monophosphate) signaling pathway: The elevation of cAMP leads to diverse cellular effects, such as airway smooth muscle relaxation, repressed effects on cellular


Introduction
An initial outbreak of pneumonia caused by unknown etiology was rst reported at Wuhan in Hubei Province, China, and alerted to the World Health Organization (WHO) by the Wuhan Municipal Health Commission on 31 December 2019 1 . Later, the infectious disease experts detected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can rapidly transmit from person to person through interaction or respiratory droplets 2 . As a consequence of its tremendous spread in the world, WHO announced a changing level from epidemic to pandemic disease (COVID- 19) on March 11, 2020 3 .
Although the symptoms are identical to pneumonia, however, a considerable number of  infected patients showed no physical sign, thus can transmit the virus to others, as silently spread 4 .
Due to the unavailability of a reliable vaccine, clinicians are utilizing anti-viral drugs and NSAIDs as a signi cant viable option for COVID-19 patients 5 . A recent study has reported that use of NSAIDs is safe for COVID-19 treatment without exposing speci c negative side effects 6 . Though there is a lack of evidence whether combined NSAIDs treatment could worsen COVID-19 symptoms 7 , but researchers suggested that anti-in ammatory therapies might suppress the fatal cytokine storm of COVID-19 patients risk of death with the administration of NSAIDs in COVID-19 patients 9 .
Commonly, NSAIDs are used to treat diverse anti-in ammatory symptoms due to its good therapeutic e cacy 10 . However, one potential drug of interest is indomethacin which possesses both antiin ammatory and antiviral properties. Its antiviral potentiality was rst identi ed in 2006 during the outbreak of SARS-CoV 11 and subsequent attribution was also observed against SARS-CoV-2 12 . A study on canine coronavirus (in vitro) revealed that indomethacin could signi cantly suppress virus replication, thus protecting host cell from virus induced damage. Similar antiviral effect was also observed during in vivo assessment where normal anti-in ammatory dose was found very effective 12,13 . Although there are many NSAIDs which may have possible therapeutic interventions against COVID-19, lack of scienti c evidence has limited their broad application to COVID-19 patients. Hence, we aimed to identify the most potent NSAIDs and their mechanism(s) against COVID-19 through network pharmacology.
Network pharmacology can decode the mechanism(s) of drug action with an overall viewpoint , which focuses on pattern changing form "single protein target, single drug" to "multiple protein targets, multiple drugs" 14 . Currently, network pharmacology has been extensively utilized to explore multiple targets and unknown additional mechanism(s) against diverse diseases 15 . In this research, network pharmacology was applied to investigate the most potent NSAIDs and their novel mechanisms of action against COVID-19. Firstly, a total of 20 approved NSAIDs was selected via using public websites. The 20 NSAIDs and COVID-19 related genes were also identi ed using public databases. Next, the selected overlapping genes were discovered as target genes for analyzing anti-COVID-19. Finally, pathway enrichment analysis was performed to reveal the mechanism(s) of the most potent NSAIDs against COVID-19. Figure 1 shows overall work ow.

Information of NSAIDs
A total of twenty FDA approved NSAIDs was selected. Table 1 and Figure 2 display the chemical information and structure of these NSAIDs. Among the twenty NSAIDs, nineteen NSAIDs were found as active drug and one "nabumetone" was a prodrug and its metabolite form is 6-methoxy-2-naphthylacetic acid (6MNA). Figure 3 shows nabumetone oxidized into 6MNA. NSAIDs linked to the 781 genes or COVID-19 related genes By screening from two public databases (SEA and STP), a total of 781 NSAIDs related genes were identi ed (see Supplementary Table S1). The overlapping genes (228 genes) selected from the two databases were shown (see Supplementary Table S2). Figure 4 displays the result of the overlapping genes. From the PubChem database, 466 COVID-19 related genes were identi ed (see Supplementary  Table S3). The 26 overlapping genes were extracted between the 228 overlapped genes and 466 COVID-19 related genes (see Supplementary Table S4). Figure 5 shows the result of overlapping genes. Pathway enrichment analysis of overlapping genes and identi cation of signi cant genes against COVID-19 Figure 6 displays the identi ed overlapping 26 genes were linked closely to each other through utilizing STRING. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis with "Homo sapiens" mode, 26 signaling pathways from the 26 genes were revealed against COVID-19. Figure 7 shows the signaling pathways were plotted in the bubble chart through Rstudio. Table 2 provides the detailed description of the 26 signaling. Figure 8 shows that both MAPK8 and MAPK10 linked to 22 out of 26 signaling pathways, were determined as hub genes of NSAIDs against COVID-19. Coincidently, both MAPK8 and MAPK10 play major roles in all of the 22 signaling pathways by the RAS signaling pathways, suggesting that BAD (Bcl-2-associated death promoter) gene and the two hub genes (MAPK8 and MAPK10) are associated with the RAS signaling pathway against COVID-19. The interactions between NSAIDs and each gene were visualized with RStudio (see Supplementary Figure S1).
A nity binding energy score on three genes of the most potent NSAIDs against

Network construction of interactions between NSAIDs targeted genesand COVID-19related genes
The overlapping genes interactions between NSAIDs targeted genes and COVID-19 related genes were analyzed by STRING (https://string-db.org/).

Signaling pathway enrichment analysis of overlapping genes
Genes-genes interaction gure was visualized by STRING (https://string-db.org/). RStudio plotted the bubble chart of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of overlapping genes. Using RStudio, the most signi cant genes among signaling pathways and correlation of NSAIDs on the most signi cant genes were analyzed. The results suggest a hint at the unknown molecular mechanism(s) of the most potent NSAIDs against COVID-19.

Discussion
NSAIDs-genes networking analysis demonstrated that the clinical effect of NSAIDs on COVID-19 was directly related to 26 genes. The results of KEGG pathway enrichment analysis of 26 genes suggested that 26 signaling pathways were associated with the occurrence and development of the COVID-19 symptoms. The correlations of 26 signaling pathways with COVID-19 symptoms were succinctly discussed as follows. PPAR (Peroxisome Proliferator-Activated Receptor) signaling pathway: A report shows that PPARγ (Peroxisome Proliferator-Activated Receptor-gamma), PPARα (Peroxisome Proliferator-Activated Receptor-alpha), and PPARβ/δ (Peroxisome Proliferator-Activated Receptor-beta/delta) agonists have anti-in ammatory and immunomodulatory functions 16 . MAPK (Mitogen-Activated Protein Kinase) signaling pathway: The mechanisms of p38 MAPK inactivation might be a signi cant therapy against the SARS infected cells 17 . Additionally, MAPK stimulates cytokine production such as IL-10 (Interleukin 10), TNF-α (Tumor Necrosis Factor-Alpha), IL-4 (Interleukin 4), and IFN-γ (Interferon gamma) 18 . This report shows a coincidence with our suggested strategy in this study. ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) signaling pathway: ErbB signaling reduces the proin ammatory activation in cardiac cells 19  Subsequently, activation of VEGFA elevates vascular permeability and severity of endothelial damage 27 . TLR (Toll-like receptor) signaling pathway: Toll-like receptors (TLRs) play a pivotal role in the innate immune system and contribute to defend host cells by recognizing PAMPs (Pathogen-Associated Molecular Patterns) induced by various microbes 28 . The activation of TLRs triggers an array of response resulting into expression of different cytokines and chemokines, phagocytosis, and even apoptotic case activation to induce programmed cell death 29 . NOD-like receptor (NLR) signaling pathway: Nod-like receptors (NLRs) have been revealed as the major microbial signals that take part in the universal immune responses to infection, and also contribute to the prevention of infections 30 . RIG-I-like receptor (RLR) signaling pathway: RIG-I-like receptors (RLRs) play a vital role in pathogen sensor of RNA virus infection, which enhances the antiviral immunity by sensing foreign RNA 31 . IL-17 (Interleukin-17) signaling pathway: IL-17 receptor inhibitors are widely used to ameliorate the in ammatory acuteness to date. Furthermore, it is a potential target to suppress severe in ammation induced by COVID-19 32 . Fc epsilon RI signaling pathway: Fc epsilon RI interconnecting causes mast cell degranulation and synthesis of proin ammatory mediators 33 . TNF (Tumor Necrosis Factor) signaling pathway: TNF de cit is associated with dysfunctional secretion of in ammatory cytokine, leading to lung pathology and death during respiratory poxvirus infection, and thus TNF is very signi cant element for regulating in ammation 34 . Neurotrophin signaling pathway: COVID-19 causes severe brain damage and destruction of central nervous system derived from neurotrophin (Huang and Reichardt 2001). Insulin signaling pathway: Obesity-oriented insulin resistance is associated with the induction of proin ammatory macrophage, leads to in ammation of adipose tissue 37 . GnRH (Gonadotropin-Releasing Hormone) signaling pathway: Disrupted BBB (Blood Brain Barrier) by viral infection, lymphocytes (B and T cells), monocytes, and granulocytes can penetrate in the brain parenchyma which induce in ammation, resulting in dysregulation of GnRH neurons. Additionally, the in ammation of GnRH neurons inhibits GnRH transport through proin ammatory cytokines by impairing the cytoskeleton 38 . Prolactin signaling pathway: HIV (Human Immunode ciency Virus) patients have greater prolactin quantity compared to others. Besides, prolactin is regarded as a cytokine to react in immune system 39,40 . Adipocytokine signaling pathway: Adipocytokines stimulate in ammation and disrupting immune response which cause tissue damage. Adipocytokines might also induce proin ammation in RA (Rheumatoid Arthritis) patients and thus lead to the development of bone damage 41 . Oxytocin signaling pathway: Oxytocin interrupts the production of proin ammatory cytokines by inactivating of the eIF-2α-ATF4 (Eukaryotic Initiation Factor -2 alpha-Activating Transcription Factor 4) pathway 42 . Relaxin signaling pathway: Relaxin inhibitors are good therapeutic targets to suppress in ammation caused by airway dysfunction 43 . AGE-RAGE (Advanced Glycation End product -Receptor of Advanced Glycation End product) signaling pathway in diabetic complications: The binding of AGE to its receptor RAGE can trigger the cytokine production, thus, can cause tissue damages, while the blockage of AGE-RAGE can effectively curtail the in ammation 44 . Epithelial cell signaling in Helicobacter pylori infection: Helicobacter pylori interrupts T and B cell signaling to set immune system. It is apparent that COVID-19 patients with Helicobacter pylori might be vulnerable to in ammatory responses 45 .
Generally, SARS-CoV-2 invades in the lungs and throat, induces excessive in ammation, which causes the secretion of cytokines, resulting in severe complications like acute respiratory failure, pneumonia, and acute liver injury (Reyes and Peniche 2019; Nile et al. 2020). Researchers suggested that RAS is a potential route for SARS-CoV-2 induced cellular infection which may be linked to the imbalance of RAS. It was discovered that ACE-2 is the functional receptor for the SARS-CoV-2 to trigger infection in the lung alveolar epithelial cells. The internalization of virus leads to downregulate the ACE-2 on host cell surface that could cause the elevation and demotion of the angiotensin-II (AII) and angiotensin 1-7 (A 1-7 ) respectively. Such an imbalance between these angiotensins may induce deleterious effects in the lung and heart. Thus, the SARS-CoV-2 affects humans through this mechanism [49][50][51][52] . Therefore, blockade of the RAS may restore the RAS balance by reducing the deleterious effects associated with angiotensin-II 53 . Recent evidence showed that RAS inhibitors might be a promising target for relieving acute-severe pneumonia caused by the COVID-19 54 .
Interestingly, our study identi ed that the three genes (MAPK 8, MAPK 10, and BAD) are mainly associated with the RAS signaling pathway. MAPK 8 and MAPK 10 are members of the MAPK family which are the key mediators of the in ammation, vasoconstriction, and thrombosis. Besides, overwhelming heart and lung injury in COVID-19 infection might be due to the overactivation of MAPK 55 . Therefore, inactivation of these genes can also be a viable strategy for relieving COVID-19 induced organ injury. In addition, disposal of in ammatory cells by promoting the cell death can be an innovative approach to control excessive in ammation. In this regard, inhibition of the anti-apoptotic Bcl-2 gene can also be a potential target to lessen in ammation 56,57 . Our ndings also explored that MAPK8, MAPK10 and BAD genes are related to three, twelve, and two NSAIDs, respectively. During the molecular docking analysis, 6MNA, rofecoxib, and indomethacin revealed promising binding a nity along with highest docking score against MAPK8, MAPK10 and BAD genes, respectively, which indicated that the three (6MNA, Rofecoxib, and Indomethacin) NSAIDs are very potential among all others, may possibly block the RAS signaling pathway by inactivating its associated genes (MAPK8, MAPK10 and BAD), and subsequently suppress SARS-CoV-2 induced cytokine storm.
Among various NSAIDs, indomethacin is a current drug of interest to the clinicians. Primary care physicians (New York) reported that indomethacin had been prescribed to a large number of COVID-19 patients and observed quick recovery from cough, pain, and other symptoms. Such improvements and well-being bene ts were not evident in the case of ibuprofen and hydroxychloroquine implementation (Vaduganathan et al. 2020b;Little 2020). Importantly, many researchers previously reported varying degrees of antiviral activity of indomethacin against herpesvirus 60 65 , and canine coronavirus 11 . In contrast, 6MNA (active metabolite of nabumetone) and Rofecoxib are also the potential antiin ammatory drugs, but studies disclosed that they are less potent compared to the indomethacin 66,67 .
Hence, such compelling outcomes indicate that indomethacin can be considered to use alone or in combination for antiviral therapy which may assist in combating human coronavirus (SARS-CoV-2).
In summary, NSAIDs-genes network suggested that the therapeutic effect of NSAIDs on COVID-19 was associated with 26 signaling pathways. This study suggests that 6MNA, rofecoxib, and indomethacin are the most potent NSAIDs against COVID-19. The basis of this research is an understanding of how these NSAIDs (which stimulates anti-in ammatory processes against COVID-19) work on COVID-19 patients. That scienti c evidence informs the selection of NSAIDs, in turn, provides for clinical design against COVID-19. Our research suggests that the inhibition of BAD-Indomethacin with other two hub genes MAPK8-6MNA, MAPK10-Rofecoxib might play cumulative actions by inactivating the RAS signaling pathway against COVID-19. Most recently, e cacy of indomethacin against COVID-19 has been approved clinically. Our study presents that indomethacin is a potent therapeutic candidate to relieve COVID-19 symptoms, which is in line with the many previous studies. However, further clinical trial on indomethacin should be warranted in COVID-19 patients in order to slow down the progression of SARS-CoV-2 and mitigating the severity as well.       An active metabolite (6MNA) of Nabumetone (Prodrug).