A total of 161 genes were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/gene), and 109 genes were searched in DisGeNET (https://www.disgenet.org/). From these two databases, 67 overlapping genes were identified (S1). Based on this 67 gene list, pathological diseases associated with coronavirus were identified using DAVID (https://david.ncifcrf.gov/) and KEGG databases (Figure 1, Table 1).
FDA-approved treatments in accordance with coronavirus-associated diseases from KEGG database were obtained from FDA and Mayo clinic (https://www.mayoclinic.org/).. Diseases that had P-values over 0.05 were considered significant and listed in Table 1. Gene count represents the number of genes from 67 genes that are associated with the disease.
Table 1. Disease-Pathway enrichment and FDA approved treatments
KEGG ID: Term
|
Gene count
|
FDA approved treatments
|
hsa05164:Influenza A
|
14
|
Oseltamivir
|
hsa05142:Chagas disease (American
|
13
|
Benznidazole
|
trypanosomiasis)
|
|
|
hsa05152:Tuberculosis
|
13
|
Isoniazid, Rifampin
|
hsa05168:Herpes simplex infection
|
12
|
Acyclovir, Famciclovir
|
hsa05166:HTLV-I infection
|
11
|
No specific treatment
|
hsa05321:Inflammatory bowel
disease (IBD)
|
10
|
Mesalamine, Balsalzide
|
hsa05161:Hepatitis B
|
10
|
Tenofovir, Lamivudine, Entecavir
|
hsa05145:Toxoplasmosis
|
9
|
Pyrimethamine, Sulfadiazine
|
hsa05162:Measles
|
9
|
No specific treatment
|
hsa05323:Rheumatoid arthritis
|
8
|
Methotrexate, leflunomide,
hydroxychloroquine, sulfasalazine, infliximab (TNF inhibitor)
|
hsa05200:Pathways in cancer
|
8
|
Cyclosporine, glucocorticoid
|
hsa05330:Allograft rejection
|
7
|
Depends on the type of rejection,
corticosteroids
|
hsa05140:Leishmaniasis
|
7
|
Liposomal amphotericin B (IV),
miltefosine
|
hsa05133:Pertussis
|
7
|
Erythromycin
|
hsa05169:Epstein-Barr virus
infection
|
7
|
No specific treatment
|
hsa04940:Type I diabetes mellitus
|
6
|
Insulin
|
hsa05144:Malaria
|
6
|
Chloroquine, Hydroxychloroquine,
Mefloquine, Proguanil
|
hsa05320:Autoimmune thyroid
disease
|
6
|
Levothyroxine
|
hsa05146:Amoebiasis
|
6
|
Metronidazole
|
hsa05160:Hepatitis C
|
6
|
Interferon, Ribavirin
|
hsa05203:Viral carcinogenesis
|
6
|
Depends on viruses
|
hsa05020:Prion diseases
|
5
|
Anthracyclines, Amphotericin B
|
hsa05212:Pancreatic cancer
|
5
|
Capecitabine, Fluorouracil
|
hsa04932:Non-alcoholic fatty liver
disease (NAFLD)
|
5
|
No treatment
|
hsa05143:African trypanosomiasis
|
4
|
Pentamidine, Suramin, Melarsoprol,
Eflornithine, Nifurtimox
|
hsa05014:Amyotrophic lateral
sclerosis (ALS)
|
4
|
Edaravone, Riluzole
|
hsa05150:Staphylococcus aureus
infection
|
4
|
Cefazolin, Cefuroxime, Oxacillin,
Vancomycin, Rifampin
|
hsa05416:Viral myocarditis
|
4
|
Enalapril, Lisinopril
|
hsa05210:Colorectal cancer
|
4
|
5-Fluorouracil (5-FU) Capecitabine
(Xeloda)
|
hsa05120:Epithelial cell signaling in
Helicobacter pylori infection
|
4
|
Omeprazole, Amoxicillin,
Clarithromycin
|
hsa05220:Chronic myeloid leukemia
|
4
|
Imatinib
|
hsa05410:Hypertrophic
cardiomyopathy (HCM)
|
4
|
beta blockers, diuretics,
Disopyramide
|
hsa05132:Salmonella infection
|
4
|
Ciprofloxacin, Azithromycin,
Ceftriaxone
|
hsa05322:Systemic lupus
erythematosus
|
4
|
Belimumab
|
hsa04614:Renin-angiotensin system
|
3
|
angiotensin-converting enzyme (ACE) inhibitors, selective AT1
receptor blockers (ARBs)
|
hsa05310:Asthma
|
3
|
Albuterol, Levalbuterol
|
hsa04930:Type II diabetes mellitus
|
3
|
Metformin, Sulfonylureas, DPP4
inhibitors
|
Interestingly, several drugs, including oseltamivir, interferon and corticosteroids, have been used in patients with coronavirus (SARS or MERS), a finding in accord with our in silico results13. Moreover, a recent study on COVID–19 reported that chloroquine or hydroxychloroquine (for malaria), which is ranked in our disease results, showed antiviral efficiency in vitro14. In the case of recent studies on biomarkers, angiotensin-converting enzyme 2 (ACE2) has received attention as a new biomarker of SARS and COVID–19, a finding in agreement with treatments for viral myocarditis and Renin-angiotensin system both of which use ACE inhibitors, such as enalapril and lisinopril as treatments. Therefore, coronavirus-associated disease-pathways and FDA-approved drug list were proposed for drug repurposing candidates and further examined to uncover molecular mechanisms in host infection against COVID–194.
Based on retrieved 67 genes, protein-protein interaction (PPI) network was retrieved from STRING database and visualized using Cytoscape (Figure 2, Figure 3). The node size reflects the number of interactions (degree) between proteins, and proteins with high number of degree were considered potent targets in our study.
From our in silico PPI network, 51 proteins from 67 genes lists were identified to be connected to form a network (S2). The proteins that showed the highest degree of interaction among all proteins was IL–6, followed by TNF, which can be considered potential targets for future coronavirus studies. Among these targets, DDP4 was recently reported to be a functional receptor for the MERS virus, a finding in agreement with our in silico results3. We also identified ACE2 that has also received attention as a new biomarker of SARS and COVID–19, as confirmed by our search 4.
Lastly, we applied the systematic dynamics that can be investigated through drug-drug network to observe coronavirus targets associated within the predicted drug combinations.
Based on the disease and FDA-approved drug list (Table 1), we selected four diseases (influenza A, Malaria, Hepatitis C, and viral myocarditis), and matching drugs (oseltamivir, hydroxychloroquine, ribavirin, and lisinopril) that are currently used in clinic or within study for COVID–19 to construct drug-drug networks (Figure 4, Figure 5).
Total six networks were constructed, and only four networks were interconnected to each other (Figure 5 a-d). Figure 5-a, Hydroxychloroquine + Ribavirin combination had the highest number of overlapping targets compared to PPI target lists (5 targets: IL6, IL2, IL10,
CASP3, IFNA1), followed by Figure 5-b Hydroxychloroquine + Lisinopril (4 targets: IL6, CASP3, ACE, ACE2), Figure 5-c Hydroxychloroquine + Oseltamivir (3 targets: IL6, TNF, CASP3), and Figure 5-d Oseltamivir+ Lisinopril (2 targets: ACE, ACE2). From these four networks, we identified two targets, IL6 and CASP3 were found in common. Overall, our result indicates that drug combination of Hydroxychloroquine + Ribavirin (Figure 5-a) is most favorable combination among six networks, and two targets IL6 and CASP3 can be potent targets for coronavirus.
Collectively, our study provides new insights in drug screening, which can be applied to further coronavirus drug development. Through in silico study, we suggest drug repurposing, uncover molecular mechanism of drugs and targets involved with coronavirus, and synergistic combination through network pharmacology. To our knowledge, this concept of drug repurposing and network pharmacology-based drug combination strategy has not yet been established elsewhere. Because of its prospective advantages of time- and cost-savings, in silico screening and drug combinations for drug repurposing can be considered a viable approach for discovering novel coronavirus treatment.