Pilaralisib inhibits the replication of enteroviruses by targeting the PI3K/AKT signaling pathway

Figures

• 

  Fig. 1. Pilaralisib efficiently inhibits EV71 replication. Chemical structure of Pilaralisib. (A). The presence of CPE was observed by light microscopy 48 h after infection, and it was found that Pilaralisib can significantly inhibit the CPE by EV71 infection (B). RD (C) and HeLa (D) cells were treated with Pilaralisib for 48 h, and an MTT assay was performed to evaluate cytotoxicity. The half-cytotoxic concentrations (CC50) of Pilaralisib in RD and HeLa cells were 14,828 nM and 23,987 nM, respectively (C–D). All experiments were independently repeated three times. The quantitative data for the screen of 1200 compounds are shown in Fig. 1E through a screening schematic (Fig. 1E)

• 

  Fig. 2. Pilaralisib exhibits antiviral effects against EV71 in vitro. RD cells were infected with EV71 (MOI = 0.1) for 2 h, followed by treatment with serially diluted Pilaralisib for 20 h at 37 °C. EV71 mRNA levels were measured by qRT-PCR (left), and the median inhibitory concentration (IC50) was calculated based on the RNA inhibition rate, which was used to determine the selectivity index (SI) (right) (A). VP1 protein expression levels were analyzed by Western blot (B). The level of EV71 replication was evaluated by immunofluorescence assay, where red indicates double-stranded RNA (dsRNA) of EV71, and blue represents the nucleus (C). The cells and supernatant were collected and then 105-fold dilution, plaque assays were performed to detect viral replication (D). The number of infectious viral clones in the samples was quantified (E-F). The cells were collected at 12, 18, and 20 h post-treatment. Viral RNA was evaluated by Western qPCR (G)

• 

  Fig. 3. Pilaralisib efficiently inhibits enteroviral replication. The cells were infected with CVB3 (A-B), CVB4-5 (C), and CVB4-7 (D) (MOI = 0.1) for a period of 2 h. After removing the supernatant, different concentrations of Pilaralisib were added to the cells. The cells were then cultured at 37 °C for 20 h. The expression levels of viral mRNA were detected using qRT-PCR (A, C, D). The presence of viral double-stranded RNA (ds-RNA) was detected using an immunofluorescence assay; red signal represents ds-RNA, blue represents the nucleus (B). The results were considered statistically significant at p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***)

• 

  Fig. 4. Effects of Pilaralisib on EV71 infection in suckling mice. The intraperitoneal inoculation of 3-day old ICR suckling mice with EV71 (8 × 106 PFU) was utilized as the experimental model. The infected suckling mice were treated with Pilaralisib on a daily basis (A). The Kaplan–Meier survival curve of mice in all groups were calculated using GraphPad prism 8 (B). To observe the pathological damage, different tissues from the mice were collected at 2 dpi and stained with HE to observe pathological damage (C). The virus was titrated by TCID50 (D). The results were considered statistically significant at p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***)

• 

  Fig. 5. Pilaralisib inhibits EV71 replication by blocking PI3K/AKT signaling pathway. RD cells were infected with EV71 at MOI of 0.01, 0.1, 1, or 10 for 2 h. Then the suspension was removed and the cells were incubated by fresh medium for 20 h. The VP1, PI3K/P-PI3K, AKT/P-AKT, and β-actin proteins were analyzed by Western blot (A). RD cells were infected with EV71 (MOI = 0.1) for 2 h. The cells were subsequently collected in four time points: 8, 12, 18, 20 h post-infection, and the VP1, PI3K/P-PI3K, AKT/P-AKT, and β-actin proteins were subjected to analysis via Western blot (B). RD cells were infected with EV71 (MOI = 0.1) for 2 h. The cells were subsequently treated with varying concentrations of Pilaralisib for 20 h. The VP1, AKT/P-AKT, and β-actin proteins were examined by Western blot (C)

• 

  Fig. 6. Pilaralisib mainly inhibits EV71 during post-entry phase. RD cells were infected with EV71 (MOI = 0.1) for 2 h at 4 °C, the cells were washed and further cultured with or without Pilaralisib for 2 h at 37 °C The viral RNA levels were tested using qPCR (A). RD cells were incubated with serially diluted Pilaralisib and EV71(MOI = 0.1) for 2 h, The expression levels of viral mRNA were detected using qRT-PCR (B)