A Transcriptomics-Based Bioinformatics Approach for Identification and In Vitro Screening of FDA-Approved Drugs for Repurposing against Dengue Virus-2

Abstract

The rising incidence of dengue virus (DENV) infections in the tropical and sub-tropical regions of the world emphasizes the need to identify effective therapeutic drugs against the disease. Repurposing of drugs has emerged as a novel concept to combat pathogens. In this study, we employed a transcriptomics-based bioinformatics approach for drug identification against DENV. Gene expression omnibus datasets from patients with different grades of dengue disease severity and healthy controls were used to identify differentially expressed genes in dengue cases, which were then applied to the query tool of Connectivity Map to identify the inverse gene-disease-drug relationship. A total of sixteen identified drugs were investigated for their prophylactic, virucidal, and therapeutic effects against DENV. Focus-forming unit assay and quantitative RT-PCR were used to evaluate the antiviral activity. Results revealed that five compounds, viz., resveratrol, doxorubicin, lomibuvir, elvitegravir, and enalaprilat, have significant anti-DENV activity. Further, molecular docking studies showed that these drugs can interact with a variety of protein targets of DENV, including the glycoprotein, the NS5 RdRp, NS2B-NS3 protease, and NS5 methyltransferase The in vitro and in silico results, therefore, reveal that these drugs have the ability to decrease DENV-2 production, suggesting that these drugs or their derivatives could be attempted as therapeutic agents against DENV infections.

Keywords: DENV-2; FDA-approved drugs; antiviral therapy; dengue fever; repurposing drugs.

Figure 1

In silico and in vitro methods used for selecting and studying the antiviral activity of drugs for repurposing.

Figure 2

Snapshot of the heat map generated by querying the DHF DEGs in CMap. The blue coloured gradient indicates the negative connectivity score of the compounds with inverse gene signatures.

Figure 3

Antiviral screening of drugs for repurposing at maximal nontoxic concentration against DENV under (a) pre-, (b) post-, and (c) co-treatment conditions. Vero CCL-81 cells were treated with a maximum non-toxic dose of drugs for 24 h in a pre-, co-, and post-infection manner and incubated for 120 h with DENV. Post incubation, plates were frozen and culture filtrates were used for different assays. Experiments were performed in triplicate in two independent trials. Results were plotted based on mean log10 of focus-forming unit/mL ± standard error. All the treatment groups were compared with a control group (VC) (infected cells without treatment). **** p < 0.0001, * p < 0.05.

Figure 4

Antiviral effects of resveratrol, lomibuvir, and elvitegravir against DENV under pre-treatment condition. Vero CCL-81 cells were treated with different concentrations of respective drugs for 24 hrs and infected with DENV-2 and incubated for 120 h. The cultured filtrates were used for the FFU assay: (a)—resveratrol, (b)—lomibuvir, and (c)—elvitegravir. Experiments were performed in triplicate at three independent time points. Results were plotted based on mean log₁₀ of focus-forming unit/mL ± standard error. All the treatment groups were compared with a virus control (VC) group (infected cells which did not receive drugs). **** p < 0.0001, *** p < 0.0005, ** p < 0.01.

Figure 5

Antiviral effects of doxorubicin, resveratrol, and enalaprilat against DENV under post-treatment conditions. Vero CCL-81 cells were administered the drug at different concentrations immediately after infection and incubated for 120 h after infection. The cultured filtrates were used for the FFU assay: (a)—doxorubicin, (b)—resveratrol, and (c)—enalaprilat. Experiments were performed in triplicate at three independent time points. Results were plotted based on mean log10 of focus-forming unit/mL ± standard error. All the treatment groups were compared with a virus control (VC) group (infected cells which did not receive drugs). **** p < 0.0001, *** p < 0.0005.

Figure 6

Antiviral effects of doxorubicin under co-treatment condition. The virus was treated with doxorubicin at different concentrations and the virus drug mixture was used for infection. Infected Vero CCL-81 cells were incubated for 120 h after infection. The culture filtrates were used for the FFU assay. Experiments were performed in triplicate at three independent time points. Results were plotted as mean log10 of focus-forming unit/mL ± standard error. All the treatment groups were compared with a virus control (VC) group (infected cells which did not receive drugs). **** p < 0.0001.

Figure 7

Effect of various drugs on the percent cell viability of the infected cells under different treatment conditions. (a)—pre-treatment, (b)—post-treatment, and (c)—co-treatment. **** p < 0.0001, *** p ≤ 0.0005, ** p < 0.01, * p < 0.05.

Figure 8

Molecular docking interaction of five repurposed drugs with DENV structural and non-structural proteins. Ribbon diagram with the solvent surface rendered view (probe radius 1.4 Å) and 2-dimensional interaction diagram showing interaction with DENV: (a) NS5 methyltransferase domain, (b) NS2B-NS3 protease domain, (c) NS5 RdRp domain, (d) NS5 RdRp domain, and (e) Envelope glycoprotein complex. (i)—3D interaction diagram, (ii)—2D interaction diagram.