Inhibition of Enterovirus A71 by a Novel 2-Phenyl-Benzimidazole Derivative

Abstract

Enterovirus A71 (EV-A71) infection has emerged as a significant public health concern atthe global level. Epidemic events of EV-A71 have been reported worldwide, and this succession of outbreaks has heightened concern that EV-A71 may become a public health threat. In recent years, widespread A71 enterovirus also occurred in European countries. EV-A71 infection causes hand-foot-mouth disease (HFMD), herpangina, and fever. However, it can sometimes induce a variety of neurological complications, including encephalitis, aseptic meningitis, pulmonary edema, and acute flaccid paralysis. We identified new benzimidazole derivatives and described their in vitro cytotoxicity and broad-spectrum anti-enterovirus activity. Among them, derivative 2b resulted in interesting activity against EV-A71, and therefore it was selected for further investigations. Compound 2b proved to be able to protect cell monolayers from EV-A71-induced cytopathogenicity, with an EC₅₀ of 3 µM. Moreover, Vero-76 cells resulted in being significantly protected from necrosis and apoptosis when treated with 2b at 20 and 80 µM. Compound 2b reduced viral adsorption to Vero-76 cells, and when evaluated in a time-of-addition assay, the derivative had the highest effect when added during the infection period. Moreover, derivative 2b reduced viral penetration into host cells. Besides, 2b did not affect intestinal monolayers permeability, showing no toxic effects. A detailed insight into the efficacy of compound 2b against EV-A71 showed a dose-dependent reduction in the viral titer, also at low concentrations. Mechanism of action investigations suggested that our derivative can inhibit viral endocytosis by reducing viral attachment to and penetration into host cells. Pharmacokinetic and toxicity predictions validated compound 2b as a good candidate for further in vivo assays.

Keywords: EV-A71; TEER; antivirals; apoptosis assay; benzimidazole derivatives; neurological complications; penetration assay; timecourse.

Scheme 1

1a,b-5a,b.

Figure 1

Effect of 2b inhibitor (20 and 80 µM) on the Vero-76-infected monolayers. Control (A), treated cells with 20 µM (B), treated cells with 80 µM (C), infected cells (D), infected treated cells (20 µM) (E), and infected treated cells (80 µM) (F). Pictures of cell morphology were taken at 72 h post-infection using ZOE Fluorescent cell imager (Bio-Rad) (bar size = 100 μm, magnification, 20×).

Figure 2

The inhibitory effect of compound 2b on EV-A71-induced apoptosis. The percentage of live, apoptotic, and necrotic cells were measured by flow cytometry using the PI-annexin V assay. Dot plots show cell death in Vero-76 cells: control (A), treated cells with 20 µM (B), treated cells with 80 µM (C), infected cells (D), infected treated cells (20 µM) (E), and infected treated cells (80 µM) (F). Percentage of live, apoptotic, and necrotic cells (G). Statistically significant differences are expressed as follows: * = p< 0.05 vs. untreated cells; *** = p< 0.001 vs. untreated cells; # = p< 0.05 vs. cells treated (20 µM) + virus; ## = p< 0.01 vs. cells treated (20 µM) + virus.

Figure 3

Measurement of cell monolayer permeabilization (transepithelial electrical resistance (TEER)) assay. Caco-2 cell monolayers were incubated with OXY at 60 µM (black squares) as negative control, derivative 2b at 20 µM (blue triangles), and Control (red circles) as internal positive control. Statistically significant differences are expressed as follows: *** = p < 0.001 vs. Control. Each value represents the mean ± SD of independent experiments (n = 3).

Figure 4

(A) Yield of infectious EV-A71 viruses produced in infected Vero-76 cells treated with 2b and NM 107. Vero-76 cells were infected with EV-A71 (m.o.i. 0.1). The infected cultures were treated with 2b, at indicated doses. Viral yields in the culture supernatant were determined by a plaque assay at 96 h post-infection. * Statistically significant differences are expressed (p< 0.05). (B) Virucidal effect (expressed as plaque-forming units (PFU/mL) of derivative 2b (20 µM) against EV-A71 virions at either 4 °C or 37 °C for 1 h. Dark columns, viral titer for viral and derivative 2b solution; white columns for the viral titer of the untreated solution. (C) Adsorption assay and time course experiment. Vero-76 cells were pre-adsorbed for 1 h at 4 °C with viruses at an m.o.i. = 1 in the presence of the compound (5 × EC₅₀ concentration). Vero-76 cells were inoculated with EV-A71 (m.o.i. = 1) and then compound 2b (20 µM) was added at the indicated times. Viral yields were determined by a plaque assay. Dark columns, the viral yield for control cells; gray columns; viral yield for cells treated with 2b derivative. (D) Penetration assay. Dose-dependency of derivative 2b (dark columns) in reduction in EV-A71 penetration compared to non-treated sample control (gray columns). The results presented were obtained from three independent experiments. Data are mean ± SD.