Antiviral activity of micafungin against enterovirus 71

Abstract

Background: Enterovirus 71 (EV71) is a major causative agent of hand-foot-mouth disease (HFMD) and also causes severe neurological complications, leading to fatality in young children. However, no effective therapy is currently available for the treatment of this infection.

Methods: We identified small-molecule inhibitors of EV71 from a screen of 968 Food and Drug Administration (FDA)-approved drugs, with which clinical application for EV71-associated diseases would be more feasible, using EV71 subgenomic replicon system. Primary hits were extensively evaluated for their antiviral activities in EV71-infected cells.

Results: We identified micafungin, an echinocandin antifungal drug, as a novel inhibitor of EV71. Micafungin potently inhibits the proliferation of EV71 as well as the replication of EV71 replicon in cells with a low micromolar IC50 (~5 μM). The strong antiviral effect of micafungin on EV71 replicon and the result from time-of-addition experiment demonstrated a targeting of micafungin on virion-independent intracellular process(es) during EV71 infection. Moreover, an extensive analysis excluded the involvement of 2C and 3A proteins, IRES-dependent translation, and also that of polyprotein processing in the antiviral effect of micafungin.

Conclusions: Our research revealed a new indication of micafungin as an effective inhibitor of EV71, which is the first case reporting antiviral activity of micafungin, an antifungal drug.

Keywords: Antiviral drug; Enterovirus; Enterovirus 71 (EV71); FDA-approved drug; Micafungin.

Fig. 1

Identification of micafungin as an anti-EV71 inhibitor from a screen of the FDA-approved drug library. a Schematic diagram of DNA encoding the EV71 replicon. b Vero cells were transfected with in vitro-transcribed EV71-replicon RNAs, immediately treated with 968 FDA-approved drugs (10 μM) for 8 h, and then assayed for firefly luciferase activity. Rupintrivir (10 μM) was used as a positive control. The luciferase activities from cells treated with 21 primary hits including micafungin were presented in graph. The luciferase activity from DMSO-treated cells was considered to be 100 %. c The antiviral activities of the primary hits were further evaluated in EV71-infected LLC-MK2 Derivative cells. The LLC-MK2 Derivative cells were infected with EV71 (100 CCID₅₀), simultaneously treated with the 21 primary hits (2 and 10 μM) for 96 h, and then cell viabilities were analyzed by using MTT assay. Rupintrivir (2 and 10 μM) was used as a positive control. The viability of DMSO-treated cells was considered to be 0 %, and that of uninfected cells was considered to be 100 %. d The chemical structure of micafungin

Fig. 2

Micafungin potently inhibits the replication of the EV71 replicon. a Vero cells were transfected with in vitro-transcribed EV71-replicon RNAs, simultaneously treated with the indicated concentrations of micafungin for 8 h, and then assayed for firefly luciferase activity. The luciferase activity of DMSO-treated cells was considered to be 100 %. b In the same condition, another set of EV71 replicon-transfected cells was assayed for cell viability using CellTiter-Glo reagent. The activity of DMSO-treated cells was considered to be 100 %

Fig. 3

Micafungin potently inhibits EV71 proliferation in LLC-MK2 Derivative cells. a LLC-MK2 Derivative cells were infected with EV71 (100 CCID₅₀) and immediately treated with increasing concentrations of micafungin. Four days after treatment, antiviral activity was determined by the reduction of the cytopathic effect in an MTT assay. Cell viability of DMSO-treated cells was set to 0 % and that of uninfected cells was set 100 %. b Same cells treated with indicated concentrations of micafungin without EV71 infection were also analyzed for cell viability by using MTT assay. c-e LLC-MK2 Derivative cells were infected with EV71 (1 MOI) and simultaneously treated with increasing concentrations of micafungin. c Twenty hours post-infection, total cell extracts were prepared from cells and subjected to Western blot analysis with anti-3C antibody. β-actin was also analyzed as a loading control. d Total RNAs were prepared from cells in (c) and then subjected to RT-PCR for 3BC region of EV71 viral RNA. β-actin mRNAs were also analyzed as a negative control. e Twenty hours post-infection, dsRNAs were stained by using specific antibody and visualized by FITC-conjugated secondary antibody (green). Nuclear DNA was also visualized by DAPI staining (blue)

Fig. 4

Antiviral activity of micafungin depending on the time of addition. LLC-MK2 Derivative cells were infected with EV71 at 1 MOI and treated with 20 μM of micafungin or 4 μM rupintrivir at the indicated times prior to or after virus infection. Twenty hours post-infection, virus-infected cells were visualized by staining with anti-dsRNA antibody and percentage of infected cells out of total cells were calculated

Fig. 5

Anti-EV71 effect of micafungin is not related with IRES-dependent translation, polyprotein processing, and 2C and 3A. a 293 T cells were transfected with dual luciferase reporter DNA measuring EV71 IRES-dependent translation and then treated with indicated concentrations of micafungin. Twenty-four hours after compound treatment cells were assayed for firefly and renilla luciferases. Luciferase activities from DMSO-treated cells were set to 100 %. b 293 T cells were transfected with plasmid expressing flag-EV71(3CD) and then treated with 10 μM of micafungin. Nine hours after compound treatment total cell extracts were prepared and subjected to Western blot analysis with anti-flag antibody. Rupintrivir (10 μM) was included as a positive control. c Vero cells were transfected with in vitro transcribed CVB3-wt, CVB3-2C mt, or CVB3-3A mt replicon RNAs and simultaneously treated with indicated concentrations of micafungin. Eight hours after compound treatment cells were assayed for luciferase activity. Luciferase activities from DMSO-treated cells were set to 100 % for each replicons