Antiviral and anti-inflammatory effects of Tabamide A derivative, TA25, against human rhinovirus and multiple zoonotic viruses in vitro and in silico

Figures

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  Fig. 1. Antiviral activity of post-, pre-, and co-treatment of TA25 against HRV-B14. Antiviral efficacy of TA25 against HRV-B14 infection in HeLa cells after post-treatment, pre-treatment, and co-treatment. Cells were infected with HRV-B14 at an MOI of 0.1 and treated with 30, 20, 10, and 1 μM of TA25. Supernatants for plaque assays and infected cells for RT-qPCR were harvested at 48 hpi. (A), (D), and (G) Expression levels analyses of HRV-B14 5’-UTR genes in HeLa cells using RT-qPCR under post-, pre-, and co-treatment conditions, respectively. (B), (E), and (H) Virus titres of HRV-B14 determined by plaque assays post-, pre-, and co-treatment, respectively. (C), (F), and (I) EC50 values of TA25 against HRV-B14 after post-, pre-, and co-treatment, respectively. The data presented are representative of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.00001, one-way ANOVA (Ut, Untreated), (ns, Not-significant).

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  Fig. 2. Down-regulation of innate immune and pro-inflammatory genes after TA25 treatment of HRV-B14 infected HeLa cells. To analyze TA25’s effect on innate immune and pro-inflammatory response genes produced by HRV-B14 infection, the changes in expression levels were compared between HRV-B14-infected cells and cells treated with 30 μM TA25 2 h after HRV-B14 infection and cells were incubated for 48 h. Total RNA-seq was performed using NovaSeq 6000, and DEGs were confirmed by RT-qPCR. (A) Heatmap of innate immune response genes in HRV-B14-infected and TA25-treated cells. (B) Heatmap of pro-inflammatory response genes in HRV-B14-infected and TA25-treated cells. (C) RT-qPCR analysis of IFN-related pathway genes and (D) pro-inflammatory genes in HRV-B14-infected and TA25-treated cells. The data presented are representative of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.00001, t-tests (Ut, Untreated), (ns, Not-significant).

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  Fig. 3. Replication of human rhinovirus is inhibited in TA25-treated cells. HRV-B14 strand-specific RT-qPCR was performed at different hpi to determine when the viral genome decreased in size during the viral life cycle. HeLa cells were treated with 30 μM of TA25 for 2 h after HRV-B14 infection at an MOI of 0.1. (A) genomic RNA and (B) anti-genomic RNA expression levels of HRV-B14 at 6, 12, 24, and 48 hpi were measured by RT-qPCR. The data presented are representative of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.00001, t-tests (Ut, Untreated), (ns, Not-significant).

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  Fig. 4. AI-driven structure-based docking analysis of TA25 and 2B protein of HRV-B14. To identify the interactions between TA25 and HRV protein, an AI-driven structure-based analysis was performed. Key residues of the 2B protein predicted to interact with TA25 were indicated (left). Electrostatic surface views were shown (middle and right). (A) The predicted pose of TA25 with the HRV 2B protein was identified computationally. (B) The predicted binding energy and affinity for TA25 binding to 2 B protein were calculated using the AI-Dock-Plus module and DeepCalici-Plus modules, respectively, in Pharmaco-Net.

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  Fig. 5. Antiviral efficacy of TA25 against multiple zoonotic viruses. To evaluate the broad-spectrum antiviral activity of TA25, its antiviral activity was tested against ZIKV, DENV, VACV, IBV Victoria and RSV. 2 h after infection, the cells were treated with 30, 20, 10, and 1 μM of TA25 and total RNA was collected at 48 hpi. The expression of (A) HRV-1A VP1, (B) ZIKV NS1, (C) DENV E, (D) VACV E9, (E) IBV Victoria HA, and (F) RSV N genes was analyzed in HeLa cells. HeLa cells were infected with HRV-1A; Vero E6 cells were infected with ZIKV, DENV, or VACV; MDCK cells were infected with IBV Victoria; and Hep-2 cells were infected with RSV. The data presented are representative of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.00001, t-tests (Ut, untreated), (ns, not-significant).