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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

Inhibition by CW-33 on EV-A71 mediated cleavage of 2A protease-specific substrates. For in vitro inhibitory enzymatic assay of r2A protease (A,B), purified r2A protease at 5 µg/mL were added to substrate (10 µg/mL) and forthwith mixed with indicated concentrations of CW-33 for 2 h at 37 °C. Mixtures developed with ABTS/H2O2 were measured at OD405, percentage inhibition of r2A protease activity calculated. *** p value < 0.001 by Scheffe test.
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viruses-07-02764-f008: Inhibition by CW-33 on EV-A71 mediated cleavage of 2A protease-specific substrates. For in vitro inhibitory enzymatic assay of r2A protease (A,B), purified r2A protease at 5 µg/mL were added to substrate (10 µg/mL) and forthwith mixed with indicated concentrations of CW-33 for 2 h at 37 °C. Mixtures developed with ABTS/H2O2 were measured at OD405, percentage inhibition of r2A protease activity calculated. *** p value < 0.001 by Scheffe test.

Mentions: With EV-A71 2A protease inhibiting Type I IFN response [11,27], interaction of CW-33 with 2A protease was predicted and analyzed by molecular docking. After global energy optimization, CW-33 was docked into active site of 2A protease consisting of Asn19, His21, Asp39, Tyr90, Gly108, Asp109, Cys110, and Ser125. Modeling of CW-33 and 2A protease had a LibDockScore of 99.6683. Figure 7 showed CW-33 interacting with 2A protease through hydrogen bonding to Gly108 and Asp109 as well as Van der Waals forming among Leu22, Val84, Ala86, Ser87, Tyr89, Tyr90, Ser105, Glu106, Gly108, Asp109, Cys110, and Ser125. These modeling interactions implied that CW-33 bound well to the active site of EV-71A 2A proteinase. To confirm the specific interaction between CW-33 and EV-A71 2A protease, inhibitory effect of CW-33 on the enzymatic activity of 2A protease was tested by in vitro cleavage assay with recombinant 2A (r2A) protease (Figure 8). In vitro cleavage assay indicated EV-A71 r2A protease significantly cleaving the substrate. Yet, CW-33 exhibited a concentration-dependent relationship with an increase of remaining substrate, revealing dose-dependent inhibition of r2A protease activity with IC50 of 53.1 μM (Figure 8A,B). In vitro cleavage of r2A protease assays revealed CW-33 manifesting an inhibitory effect on EV-A71 2A protease activity in dose-dependent manners. Results confirmed CW-33 specifically binding to the active site of EV-A71 2A protease.


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)

Inhibition by CW-33 on EV-A71 mediated cleavage of 2A protease-specific substrates. For in vitro inhibitory enzymatic assay of r2A protease (A,B), purified r2A protease at 5 µg/mL were added to substrate (10 µg/mL) and forthwith mixed with indicated concentrations of CW-33 for 2 h at 37 °C. Mixtures developed with ABTS/H2O2 were measured at OD405, percentage inhibition of r2A protease activity calculated. *** 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-f008: Inhibition by CW-33 on EV-A71 mediated cleavage of 2A protease-specific substrates. For in vitro inhibitory enzymatic assay of r2A protease (A,B), purified r2A protease at 5 µg/mL were added to substrate (10 µg/mL) and forthwith mixed with indicated concentrations of CW-33 for 2 h at 37 °C. Mixtures developed with ABTS/H2O2 were measured at OD405, percentage inhibition of r2A protease activity calculated. *** p value < 0.001 by Scheffe test.
Mentions: With EV-A71 2A protease inhibiting Type I IFN response [11,27], interaction of CW-33 with 2A protease was predicted and analyzed by molecular docking. After global energy optimization, CW-33 was docked into active site of 2A protease consisting of Asn19, His21, Asp39, Tyr90, Gly108, Asp109, Cys110, and Ser125. Modeling of CW-33 and 2A protease had a LibDockScore of 99.6683. Figure 7 showed CW-33 interacting with 2A protease through hydrogen bonding to Gly108 and Asp109 as well as Van der Waals forming among Leu22, Val84, Ala86, Ser87, Tyr89, Tyr90, Ser105, Glu106, Gly108, Asp109, Cys110, and Ser125. These modeling interactions implied that CW-33 bound well to the active site of EV-71A 2A proteinase. To confirm the specific interaction between CW-33 and EV-A71 2A protease, inhibitory effect of CW-33 on the enzymatic activity of 2A protease was tested by in vitro cleavage assay with recombinant 2A (r2A) protease (Figure 8). In vitro cleavage assay indicated EV-A71 r2A protease significantly cleaving the substrate. Yet, CW-33 exhibited a concentration-dependent relationship with an increase of remaining substrate, revealing dose-dependent inhibition of r2A protease activity with IC50 of 53.1 μM (Figure 8A,B). In vitro cleavage of r2A protease assays revealed CW-33 manifesting an inhibitory effect on EV-A71 2A protease activity in dose-dependent manners. Results confirmed CW-33 specifically binding to the active site of EV-A71 2A protease.

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