Discovery of gramine derivatives that inhibit the early stage of EV71 replication in vitro

Abstract

Enterovirus 71 (EV71) is a notable causative agent of hand, foot, and mouth disease in children, which is associated with an increased incidence of severe neurological disease and death, yet there is no specific treatment or vaccine for EV71 infections. In this study, the antiviral activity of gramine and 21 gramine derivatives against EV71 was investigated in cell-based assays. Eighteen derivatives displayed some degree of inhibitory effects against EV71, in that they could effectively inhibit virus-induced cytopathic effects (CPEs), but the anti-EV71 activity of the lead compound gramine was not observed. Studies on the preliminary modes of action showed that these compounds functioned by targeting the early stage of the EV71 lifecycle after viral entry, rather than inactivating the virus directly, inhibiting virus adsorption or affecting viral release from the cells. Among these derivatives, one (compound 4s) containing pyridine and benzothiazole units showed the most potency against EV71. Further studies demonstrated that derivative 4s could profoundly inhibit viral RNA replication, protein synthesis, and virus-induced apoptosis in RD cells. These results indicate that derivative 4s might be a feasible therapeutic agent against EV71 infection and that these gramine derivatives may provide promising lead scaffolds for the further design and synthesis of potential antiviral agents.

Figure 1

Gramine and its synthetic derivatives inhibited virus-induced CPE. (A) Vero cells were infected with 100 TCID₅₀ of EV71 mixed with serial dilutions of tested compounds for 1.5 h at 37 °C; the inoculum was aspirated and cells were incubated with DMEM/tested compounds at 37 °C, 5% CO₂ for 48 h pi. The viability of the cells was determined with an MTT assay; (B) Morphology image of Vero and RD cells treated with compounds 4a, 4i, 4r, and 4s for 48 h pi. (magnification, 20×).

Figure 2

Analysis of modes of action against EV71 of gramine derivatives. Analysis of effective stage. The RD cells were treated with tested compounds before, simultaneously or after EV71 (100 TCID₅₀) inoculation: antiviral effects were detected by measuring (A) cell viability and (B) progeny virus yields (10 µg/mL 4a, 20 µg/mL 4i, 40 µg/mL 4r and 4s) 48 h post infection; (C) Analysis of the effects on EV71 adsorption. Mock- or 10 µg/mL 4a, 20 µg/mL 4i, 40 µg/mL 4r and 4s-treated EV71 (10⁴ TCID₅₀) was spinoculated onto RD cells and adsorbed for 2 h; intracellular virus was subjected to titration using the TCID₅₀ method; (D) Effects on EV71 release from RD cells. RD cells infected with 100 TCID₅₀ of EV71 were incubated with 10 µg/mL 4a, 20 µg/mL 4i, 40 µg/mL 4r and 4s for 12 h, of both cells and supernatants (intra- and extracellular), or of cells or supernatants harvested separately for virus titration. The figure shows the inhibition rate of progeny virus yield compared with the virus control. Values represent the mean ± standard deviation of three independent experiments.

Figure 3

Time-of-addition assay. 10 µg/mL 4a, 20 µg/mL 4i, or 40 µg/mL 4r or 4s were added to RD cells at different time periods after EV71 infection. At 12 h pi, the progeny virus yield was determined (−1–0 h: viral infection period; 0–10 h: period for virus proliferation in the cells). Values are represented as the mean ± standard deviation.

Figure 4

The effect of 4s on EV71 replication in RD cells. RD cells infected with 100 TCID₅₀ of EV71 were incubated in the absence (virus control) or presence of 40 µg/mL 4s and harvested at the indicated times pi. (A) Total RNA was extracted from cells and culture supernatants and EV71 RNA levels were measured. Cellular actin amplification was used for normalization. The ΔΔCt data were calculated from three independent experiments; (B) EV71-protein was determined by indirect immunofluorescence using a mouse anti-enterovirus 71 monoclonal antibody and an Alexa-Fluor-488-conjugated AffiniPure goat anti-mouse IgG (H + L). The nucleus was stained with DAPI; the green foci indicate the presence of EV71 protein.

Figure 5

Inhibition effect of 4s on EV71-induced apoptosis. RD cells were (A) left untreated or (B) infected with 100 TCID₅₀ of EV71. After viral adsorption, RD cells were incubated in the (B) absence or (C) presence of 40 µg/mL 4s for 36–48 h; (D) The cells were stained with Annexin-V- fluorescein and propidium iodide and measured using flow cytometry.