PLX8394, a RAF inhibitor, inhibits enterovirus 71 replication by blocking RAF/MEK/ERK signaling

Fig. 1. Discovery of potent and low-toxicity active compounds that block EV71 infection. A Structure of PLX8394. B After infection with EV71 (MOI ​= ​0.1) for 2 ​h, infected-RD cells were treated with PLX8394 for 48 ​h. Images of the CPE were acquired using an optical microscope. C–F RD or HeLa cells were incubated with serially diluted PLX8394 or ribavirin for 48 ​h. Cytotoxicity was assessed using the MTT assay. CC50 values of PLX8394 in RD and HeLa cells were 19,075 and 37,523 ​nmol/L, respectively (C–D). CC50 values of ribavirin in RD and HeLa cells were 461,000 and 123,100 ​nmol/L, respectively (E–F). All experiments were independently repeated three times.

Fig. 2. Antiviral effect of PLX8394 in vitro. A–B After infection with EV71 (MOI ​= ​0.1) for 2 ​h, RD cells were treated with PLX8394 or ribavirin for 20 ​h. VP1 protein levels were examined by Western blot analysis (A). Viral RNA levels relative to GADPH levels were detected by RT-qPCR (B). C–D The treated RD cells and culture medium were freeze-thawed three times and the number of infective clones of EV71 was determined by the plaque assay (C). Quantitative data of the plaque assay (D). (E) Immunofluorescence staining evaluation of the antiviral effect of PLX8394. dsRNA of EV71 is labeled with red fluorescence (CY3), nuclei are labeled with blue fluorescence (DAPI). ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001. All experiments were independently repeated three times.

Fig. 3. PLX8394 targets EV71 at the phase of post-entry, and the inhibition can last for 20 ​h. A–C After infection with EV71 (MOI ​= ​0.1) for 2 ​h, RD cells were treated with PLX8394 for 20 ​h. RNA levels were tested by RT-qPCR. D–F RD cells were treated with serially diluted PLX8394 for 2 ​h. Then, the suspension was removed and the cells were infected with EV71 (MOI ​= ​0.1) for 2 ​h and cultured for 20 ​h. RNA levels were examined by RT-qPCR (B, E), and IC50 values were calculated as the inhibition rate of viral RNA levels, and SI values were calculated as CC50/IC50 (C, F). G–H After infection with EV71 (MOI ​= ​0.1) for 2 ​h, the infected-RD cells were treated with PLX8394 or ribavirin at a concentration of 4000 ​nmol/L for 20 ​h. VP1 protein levels at 8, 12, 16, and 20 ​h were examined by Western blot (G) and RNA levels at 8, 12, 16, and 20 ​h were examined by RT-qPCR (H). ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001. All experiments were independently repeated three times.

Fig. 4. PLX8394 inhibits EV71 replication by blocking the RAF/MEK/ERK signaling pathway. A RD cells were infected with EV71 at an MOI of 0.01, 0.1, 1, or 10 for 2 ​h. After removing the suspension, the cells were further cultured for 12 ​h. VP1, C-RAF, MEK, and ERK protein levels and the phosphorylation levels of C-RAF, MEK, and ERK were analyzed by Western blot. B After infection with EV71 (MOI ​= ​0.1) for 2 ​h, and then removal of the suspension, the cells were collected at 8, 12, 18, and 20 ​h. VP1, C-RAF, MEK, and ERK proteins levels and the phosphorylation levels of C-RAF, MEK, and ERK were analyzed by Western blot. C RD cells were treated with serially diluted PLX8394 for 20 ​h and C-RAF, MEK, and ERK proteins levels and the phosphorylation levels of C-RAF, MEK, and ERK were examined by Western blot. β-actin was used as a loading control. D After infection with EV71 (MOI ​= ​0.1) for 2 ​h, the cells were treated with different concentrations of PLX8394 for 20 ​h. VP1, C-RAF, MEK, and ERK proteins levels and the phosphorylation levels of C-RAF, MEK, and ERK were examined by Western blot. All experiments were independently repeated three times.

Fig. 5. PLX8394 exhibits broad-spectrum antiviral activity against enteroviruses. HeLa cells were infected with CVB3 (A), CVB4-5 (B), CVB4-7 (C), or ECHO11 (D) at an MOI of 0.1 for 2 ​h and then treated with serially diluted PLX8394 for 20 ​h. Viral RNA levels were detected by RT-qPCR. E The IC50 values of PLX8394 against selected enteroviruses were determined in experiments described above. ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001. All experiments were independently repeated three times.

Fig. 6. PLX8394 exerts anti-EV71 activity in vivo. A Diagram of the animal experiment. Three-day-old suckling ICR mice were infected with EV71 at 106 ​PFU and treated with PLX8394 at 5 ​mg/kg or placebo over the following 8 days. B The survival rate was monitored daily until 8 days post-infection. C–D Heart and liver tissues from mice in the “normal control,” “non-treatment,” and “treatment” groups were collected on day 2 post-infection and viral RNA levels were analyzed by RT-qPCR. E The tissues were stained with HE to reveal pathological changes. ∗P ​< ​0.05, ∗∗P ​< ​0.01, ∗∗∗P ​< ​0.001. All experiments were independently repeated three times.