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Antiviral Potential of a Novel Compound CW-33 against Enterovirus A71 via Inhibition of Viral 2A Protease.

Wang CY, Huang AC, Hour MJ, Huang SH, Kung SH, Chen CH, Chen IC, Chang YS, Lien JC, Lin CW - Viruses (2015)

Bottom Line: Molecular docking revealed CW-33 binding to EV-A71 2A protease active sites, correlating with an inhibitory effect of CW33 on in vitro enzymatic activity of recombinant 2A protease IC50 = 53.1 µM).The results demonstrated CW-33 inhibiting viral 2A protease activity to reduce Type I IFN antagonism of EV-A71.Therefore, CW-33 combined with a low-dose of Type I IFN could be applied in developing alternative approaches to treat EV-A71 infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 40402, Taiwan. yschang@mail.cmu.edu.tw.

ABSTRACT
Enterovirus A71 (EV-A71) in the Picornaviridae family causes hand-foot-and-mouth disease, aseptic meningitis, severe central nervous system disease, even death. EV-A71 2A protease cleaves Type I interferon (IFN)-α/β receptor 1 (IFNAR1) to block IFN-induced Jak/STAT signaling. This study investigated anti-EV-A7l activity and synergistic mechanism(s) of a novel furoquinoline alkaloid compound CW-33 alone and in combination with IFN-β Anti-EV-A71 activities of CW-33 alone and in combination with IFN-β were evaluated by inhibitory assays of virus-induced apoptosis, plaque formation, and virus yield. CW-33 showed antiviral activities with an IC50 of near 200 µM in EV-A71 plaque reduction and virus yield inhibition assays. While, anti-EV-A71 activities of CW-33 combined with 100 U/mL IFN-β exhibited a synergistic potency with an IC50 of approximate 1 µM in plaque reduction and virus yield inhibition assays. Molecular docking revealed CW-33 binding to EV-A71 2A protease active sites, correlating with an inhibitory effect of CW33 on in vitro enzymatic activity of recombinant 2A protease IC50 = 53.1 µM). Western blotting demonstrated CW-33 specifically inhibiting 2A protease-mediated cleavage of IFNAR1. CW-33 also recovered Type I IFN-induced Tyk2 and STAT1 phosphorylation as well as 2\',5\'-OAS upregulation in EV-A71 infected cells. The results demonstrated CW-33 inhibiting viral 2A protease activity to reduce Type I IFN antagonism of EV-A71. Therefore, CW-33 combined with a low-dose of Type I IFN could be applied in developing alternative approaches to treat EV-A71 infection.

No MeSH data available.


Related in: MedlinePlus

Inhibitory effect of CW-33 in combination with IFN-β on EV-A71-infected cytopathic effect. Firstly, cytotoxicity of CW-33 in combination with IFN-β to RD cells was performed using MTT assays (A). RD cells infected with EV-A71 were forthwith treated with CW-33 alone and in combination with IFN-β. Virus-induced cytopathicity was photographed post-infection by phase-contrast microscopy (B), apoptosis of infected and/or treated cells analyzed by flow cytometry with Annexin-V/PI staining (C). * p value < 0.05; *** p value < 0.001 by Scheffe test.
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viruses-07-02764-f004: Inhibitory effect of CW-33 in combination with IFN-β on EV-A71-infected cytopathic effect. Firstly, cytotoxicity of CW-33 in combination with IFN-β to RD cells was performed using MTT assays (A). RD cells infected with EV-A71 were forthwith treated with CW-33 alone and in combination with IFN-β. Virus-induced cytopathicity was photographed post-infection by phase-contrast microscopy (B), apoptosis of infected and/or treated cells analyzed by flow cytometry with Annexin-V/PI staining (C). * p value < 0.05; *** p value < 0.001 by Scheffe test.

Mentions: IFN-β at a low concentration (100 U/mL) slightly inhibited EV-A71-induced cytopathy (Figure 3C,D). Plaque reduction assay indicated IFN-β exhibiting less antiviral activity (IC50 = 966.2 U/mL) against EV-A71 infection. Combined treatment of serial concentrations of CW-33 with 100 U/mL IFN-β showed a more potent inhibition of virus-induced cytopathic effect and apoptosis compared to CW-33 or IFN-β alone (Figure 4vs.Figure 2). For determining additive or synergistic antiviral activity of CW-33 in combination with IFN-β, combined treatment of serial concentrations of CW-33 with 100 U/mL IFN-β was evaluated by EV-A71 plaque and yield reduction assays (Figure 5 and Figure 6). Combined treatment of CW-33 with 100 U/mL IFN-β exhibited synergistic antiviral activities against EV-A71 (IC50 of 0.9 μM for plaque reduction and IC50 of 1.4 μM for virus yield reduction). CW-33 in combination with 100 U/mL IFN-β exceeded 100-fold lower IC50 values against EV-A71 replication in vitro compared to CW-33 alone. Results demonstrated a synergistic antiviral activity of CW-33 in combination with a low concentration of IFN-β against EV-A71.


Antiviral Potential of a Novel Compound CW-33 against Enterovirus A71 via Inhibition of Viral 2A Protease.

Wang CY, Huang AC, Hour MJ, Huang SH, Kung SH, Chen CH, Chen IC, Chang YS, Lien JC, Lin CW - Viruses (2015)

Inhibitory effect of CW-33 in combination with IFN-β on EV-A71-infected cytopathic effect. Firstly, cytotoxicity of CW-33 in combination with IFN-β to RD cells was performed using MTT assays (A). RD cells infected with EV-A71 were forthwith treated with CW-33 alone and in combination with IFN-β. Virus-induced cytopathicity was photographed post-infection by phase-contrast microscopy (B), apoptosis of infected and/or treated cells analyzed by flow cytometry with Annexin-V/PI staining (C). * p value < 0.05; *** p value < 0.001 by Scheffe test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488731&req=5

viruses-07-02764-f004: Inhibitory effect of CW-33 in combination with IFN-β on EV-A71-infected cytopathic effect. Firstly, cytotoxicity of CW-33 in combination with IFN-β to RD cells was performed using MTT assays (A). RD cells infected with EV-A71 were forthwith treated with CW-33 alone and in combination with IFN-β. Virus-induced cytopathicity was photographed post-infection by phase-contrast microscopy (B), apoptosis of infected and/or treated cells analyzed by flow cytometry with Annexin-V/PI staining (C). * p value < 0.05; *** p value < 0.001 by Scheffe test.
Mentions: IFN-β at a low concentration (100 U/mL) slightly inhibited EV-A71-induced cytopathy (Figure 3C,D). Plaque reduction assay indicated IFN-β exhibiting less antiviral activity (IC50 = 966.2 U/mL) against EV-A71 infection. Combined treatment of serial concentrations of CW-33 with 100 U/mL IFN-β showed a more potent inhibition of virus-induced cytopathic effect and apoptosis compared to CW-33 or IFN-β alone (Figure 4vs.Figure 2). For determining additive or synergistic antiviral activity of CW-33 in combination with IFN-β, combined treatment of serial concentrations of CW-33 with 100 U/mL IFN-β was evaluated by EV-A71 plaque and yield reduction assays (Figure 5 and Figure 6). Combined treatment of CW-33 with 100 U/mL IFN-β exhibited synergistic antiviral activities against EV-A71 (IC50 of 0.9 μM for plaque reduction and IC50 of 1.4 μM for virus yield reduction). CW-33 in combination with 100 U/mL IFN-β exceeded 100-fold lower IC50 values against EV-A71 replication in vitro compared to CW-33 alone. Results demonstrated a synergistic antiviral activity of CW-33 in combination with a low concentration of IFN-β against EV-A71.

Bottom Line: Molecular docking revealed CW-33 binding to EV-A71 2A protease active sites, correlating with an inhibitory effect of CW33 on in vitro enzymatic activity of recombinant 2A protease IC50 = 53.1 µM).The results demonstrated CW-33 inhibiting viral 2A protease activity to reduce Type I IFN antagonism of EV-A71.Therefore, CW-33 combined with a low-dose of Type I IFN could be applied in developing alternative approaches to treat EV-A71 infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 40402, Taiwan. yschang@mail.cmu.edu.tw.

ABSTRACT
Enterovirus A71 (EV-A71) in the Picornaviridae family causes hand-foot-and-mouth disease, aseptic meningitis, severe central nervous system disease, even death. EV-A71 2A protease cleaves Type I interferon (IFN)-α/β receptor 1 (IFNAR1) to block IFN-induced Jak/STAT signaling. This study investigated anti-EV-A7l activity and synergistic mechanism(s) of a novel furoquinoline alkaloid compound CW-33 alone and in combination with IFN-β Anti-EV-A71 activities of CW-33 alone and in combination with IFN-β were evaluated by inhibitory assays of virus-induced apoptosis, plaque formation, and virus yield. CW-33 showed antiviral activities with an IC50 of near 200 µM in EV-A71 plaque reduction and virus yield inhibition assays. While, anti-EV-A71 activities of CW-33 combined with 100 U/mL IFN-β exhibited a synergistic potency with an IC50 of approximate 1 µM in plaque reduction and virus yield inhibition assays. Molecular docking revealed CW-33 binding to EV-A71 2A protease active sites, correlating with an inhibitory effect of CW33 on in vitro enzymatic activity of recombinant 2A protease IC50 = 53.1 µM). Western blotting demonstrated CW-33 specifically inhibiting 2A protease-mediated cleavage of IFNAR1. CW-33 also recovered Type I IFN-induced Tyk2 and STAT1 phosphorylation as well as 2\',5\'-OAS upregulation in EV-A71 infected cells. The results demonstrated CW-33 inhibiting viral 2A protease activity to reduce Type I IFN antagonism of EV-A71. Therefore, CW-33 combined with a low-dose of Type I IFN could be applied in developing alternative approaches to treat EV-A71 infection.

No MeSH data available.


Related in: MedlinePlus