Antiviral Activity of Compound L3 against Dengue and Zika Viruses In Vitro and In Vivo

Abstract

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne flaviviruses that cause severe illness after infection. Currently, there are no specific or effective treatments against DENV and ZIKV. Previous studies have shown that tyrosine kinase activities and signal transduction are involved in flavivirus replication, suggesting a potential therapeutic strategy for DENV and ZIKV. In this study, we found that compound L3 can significantly reduce viral protein expression and viral titers in HEK-293, MCF-7, HepG2, and Huh-7 cells and exhibits superior therapeutic efficacy against flaviviral infection compared to other tyrosine kinase inhibitors. In addition, compound L3 can decrease endogenous HER2 activation and inhibit the phosphorylation of the HER2 downstream signaling molecules Src and ERK1/2, the levels of which have been associated with viral protein expression in MCF-7 cells. Moreover, silencing HER2 diminished DENV-2 and ZIKV expression in MCF-7 cells, which suggests that HER2 activity is involved in flavivirus replication. Furthermore, in DENV-2-infected AG129 mice, treatment with compound L3 increased the survival rates and reduced the viremia levels. Overall, compound L3 demonstrates therapeutic efficacy both in vitro and in vivo and could be developed as a promising antiviral drug against emerging flaviviruses or for concurrent DENV and ZIKV outbreaks.

Keywords: Zika virus; anti-viral drugs; dengue virus; flavivirus; tyrosine kinase inhibitors.

Figure 1

Molecular structures and antiviral abilities of a series of afatinib-derivative tyrosine kinase inhibitor (TKI) compounds. (A) Schematic structures of afatinib and compounds 10b, L1, and L3. (B) HEK-293 cells were treated with a solvent or with different concentrations of afatinib and compounds 10b, L1, and L3 for 36 h. The WST-1 assay was used to measure cell viability. A percentage was obtained by comparison with the solvent control, set at 100%. (C,D) HEK-293 cells infected with dengue virus (DENV)-2 (multiplicity of infection (MOI) = 1) and treated with afatinib (C) or with compounds 10b, L1, and L3 (D) at 10 and 20 μM. After 36 h, a Western blot analysis of viral protein levels in the cell lysates was performed, and the ratio of the viral NS3 protein level to the level of actin was adjusted to that of the solvent control. Data are the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 according to a two-tailed Student’s t-test.

Figure 2

Antiviral activities of compound L3 against DENV-1, DENV-2, and ZIKV in HEK-293 cells. HEK-293 cells were infected with DENV-1, -2, or ZIKV with or without (solvent) various concentrations of compound L3 for 36 h. (A) Viral protein levels were determined by Western blot analysis. Actin or GAPDH was used as a loading control. Relative ratios of viral NS3 or E protein levels to actin or GAPDH levels were adjusted to those of the solvent control. (B) The viral progeny production in the culture supernatants was measured by a focus-forming assay. Data are the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 according to a two-tailed Student’s t-test.

Figure 3

Compound L3 significantly inhibited DENV-2 or ZIKV compared to other tyrosine kinase inhibitors. HEK-293 cells were infected with DENV-2 (A,B) or ZIKV (C,D) and treated with 10 μM of either compound L3 or the indicated drugs for 36 h. Viral protein expression (A,C) and virus titers (B,D) were analyzed and adjusted to those of the solvent control. Data are the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 according to a two-tailed Student’s t-test.

Figure 4

Compound L3 suppressed DENV and ZIKV replication through the HER2 signaling pathway. (A–C) MCF-7 cells were infected with DENV-1 for 36 h (A), DENV-2 for 30 h (B), or ZIKV for 36 h (C) (MOI = 1) and treated with 10, 20, or 40 μM of compound L3 to monitor the phosphorylation of HER2, Src, and ERK1/2 signaling molecules by Western blotting. Relative ratios of p-HER2, p-Src, and p-ERK1/2 levels to HER2, Src, and ERK1/2 levels were adjusted to those of the mock control. Viral protein levels were also determined by Western blot analysis. The relative ratios of viral NS3 or E protein levels to actin or GAPDH levels were adjusted to those of the solvent control. Actin or GAPDH was used as the loading control. (D–F) Viral titers in culture supernatants were measured by a focus-forming assay. Data are the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 by a two-tailed Student’s t-test. (G,H) MCF-7 cells were transiently transfected with an siRNA-HER2 (siHER2) or an siRNA negative control (siNC). At 24 h after transfection, the cells were infected with DENV-2 (G) or ZIKV (H) (MOI = 1) for 24 h and lysed for Western blot analysis. GAPDH was used as a loading control. The relative ratios of viral NS3 or E protein levels to GAPDH levels were adjusted to those of the solvent control.

Figure 5

Compound L3 exhibited protective efficacy in vivo. AG129 mice were infected with 10⁷ focus-forming unit (FFU) of DENV-2 and treated orally with 5 or 10 mg/kg of compound L3 daily for 7 days. (A) Survival rates were determined for 30 days. The number of animals (n) in each group is shown. The data are representative results of three independent experiments. * p < 0.05. (B) Mouse sera were collected on day 3 after treatment. A focus-forming assay was used to measure DENV-2 viremia. Data are the mean ± SD of three independent experiments. ** p < 0.01, *** p< 0.001 according to a two-tailed Student’s t-test.