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. 2023 Dec 9;24(24):17296.
doi: 10.3390/ijms242417296.

Evaluation of the Antiviral Activity of Tabamide A and Its Structural Derivatives against Influenza Virus

Soo Yong Shin  1 , Joo Hee Lee  1 , Jin Woo Kim  1 , Wonkyun Ronny Im  1 , Kongara Damodar  2 , Hyung Ryeol Woo  2 , Won-Keun Kim  3 , Jeong Tae Lee  2 , Sung Ho Jeon  1
Affiliations

Evaluation of the Antiviral Activity of Tabamide A and Its Structural Derivatives against Influenza Virus

Soo Yong Shin et al. Int J Mol Sci. .

Abstract

Influenza viruses cause severe endemic respiratory infections in both humans and animals worldwide. The emergence of drug-resistant viral strains requires the development of new influenza therapeutics. Tabamide A (TA0), a phenolic compound isolated from tobacco leaves, is known to have antiviral activity. We investigated whether synthetic TA0 and its derivatives exhibit anti-influenza virus activity. Analysis of structure-activity relationship revealed that two hydroxyl groups and a double bond between C7 and C8 in TA0 are crucial for maintaining its antiviral action. Among its derivatives, TA25 showed seven-fold higher activity than TA0. Administration of TA0 or TA25 effectively increased survival rate and reduced weight loss of virus-infected mice. TA25 appears to act early in the viral infection cycle by inhibiting viral mRNA synthesis on the template-negative strand. Thus, the anti-influenza virus activity of TA0 can be expanded by application of its synthetic derivatives, which may aid in the development of novel antiviral therapeutics.

Keywords: antiviral drugs; influenza viruses; structural derivatives; tabamide A; viral RNA synthesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Tabamide A has antiviral activity against IAV infection. MDCK cells were infected with 1 MOI of IAV and treated with the indicated amount of TA0 for 24 h. (A) Virus-induced cytopathic effects were observed under a microscope (top), and cell viability was determined by measuring the absorbance at 450 nm (bottom). (B) Relative viral M1 RNA expression following viral infection and TA0 treatment was determined by qRT- PCR. (C) The expression level of NS1 protein was measured by immunoblot analysis. All values are expressed as mean ± SD (n = 3). ### p < 0.001 compared to the cell control and *** p < 0.001 compared to the virus treated control. TA0: tabamide A; IAV: influenza A virus; MDCK: Madin–Darby canine kidney; MOI: multiplicity of infection; qRT-PCR: quantitative real-time polymerase chain reaction; NS1: nonstructural protein 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; SD: standard deviation.
Figure 2
Figure 2
Evaluation of antiviral efficacy of primary structural derivatives of TA0. (A) Structures of TA0 and its structural derivatives (TA11-TA15). Δ7,8 indicates that there is a double bond between 7 and 8. (B,C) MDCK cells were infected with 0.5 MOI of virus and treated with 100 μM of TA0 or primary derivatives. After 24 h of treatment, changes in the expression level of NS1 protein were measured by immunoblot analysis (B). The number of plaques produced 48 h after infection with the serially diluted virus was determined quantitatively (C). Infectious virus titers were expressed as PFU/mL, and results are shown as mean ± SD (n = 3). * p < 0.05 and *** p < 0.001 compared to the virus treated control (mock). TA11–15: synthetic derivatives of TA0; PFU: plaque forming unit.
Figure 3
Figure 3
Structure of secondary derivatives of TA0. Secondary derivatives of TA0 were synthesized by substituting three positions of α and β, indicated by squares in the structural diagram of TA0.
Figure 4
Figure 4
Secondary derivatives of TA0 have enhanced anti-influenza virus activity. (A) MDCK cells were infected with 0.5 MOI of IAV and treated with TA0 or its secondary derivatives at a concentration of 100 μM for 24 h. The expression level of NS1 protein was measured by immunoblot analysis. (B) The number of plaques produced by viral infection was quantitatively determined and expressed as PFU/mL. Results are expressed as mean ± SD (n = 3). (C) and IAV infection (mock), and ** p < 0.01 and *** p < 0.001 compared to the mock. (C) Expression levels of total IAV protein were examined by immunoblot analysis using an anti-IAV antibody after treatment with TA0 or its derivatives (TA23–TA25) at concentrations of 12.5 and 25 μM. TA23–25: synthetic derivatives of TA0.
Figure 5
Figure 5
Half maximal inhibitory concentration (IC50) of TA0 and its derivatives. (A) IC50 values were determined by plaque inhibition assay. MDCK cells were infected with 0.01 MOI of virus and treated with various concentrations (0.1 to 100 μM) of TA0, TA23, TA24, and TA25. After 48 h, the number of plaques was measured. Black line: graph by actual data; red dot cross line: IC50 concentration (50% inhibition/concentration) (B) IC50 of TA0 and its derivatives are listed in table (top), and a hypothetical dose–response curve based on the measured values is shown in a graph for efficacy comparison between each compound (bottom). Results are presented as mean ± SD (n = 3). Selectivity index (SI) = CC50*/IC50. In the case of cytotoxicity concentration 50% (CC50) value, the maximum concentration of 200 μM or higher in the experimental group is not the measured value, but more than the maximum treatment concentration.
Figure 6
Figure 6
Tabamide A and its derivative TA25 inhibit influenza virus infection in vivo. Mice were challenged with 2 units of LD50 (A) or sublethal dose (B) of mouse-adapted influenza virus via the intranasal route. After 1 h, TA0 or TA25 were intranasally administered and survival rate or body weight loss were observed for 14 days. Results are shown as mean ± SD (n = 5 per group in the experiment). Mock: noninfected control; IAV: virus-infected control; LD: lethal dose.
Figure 7
Figure 7
TA25 inhibits viral amplification in the early stages of viral infection. (A) A549 cells were infected with 2 MOI virus or transfected with a plasmid overexpressing the NS1 protein and treated with 20 μM TA25. The expression level of each protein was determined by immunoblot analysis using a specific anti-human protein antibody. (B) A549 cells were infected with 2 MOI virus for 1 h, and the medium containing virus was replaced and further cultured. After 4 and 8 h of infection, the relative expression of (+) strand RNA (mRNA and cRNA) or (−) strand RNA (vRNA) of the NS1 gene was measured by qRT-PCR. Results are presented as mean ± SD (n = 3). *** p < 0.001 compared to the virus treated control (mock).
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