Novel capsid binder and PI4KIIIbeta inhibitors for EV-A71 replication inhibition

Abstract

The Hand, Foot and Mouth Disease (HFMD) is a highly contagious viral illness generally manifests as a mild disease in young children and immunocompromised adults. It has however emerged as a significant public health threat in recent years as outbreaks have been occurring regularly, especially in the Asia-Pacific. The disease can result from infections by a wide variety of human enteroviruses, particularly, Enterovirus A71 (EV-A71) has garnered more attention due to its association with severe disease in infected patients. Despite the potential to result severe neurological complications or even fatality, there is currently no effective antiviral for treatment of EV-A71 infections and the only vaccines available are restricted to distribution in China. In this study, we report the in vitro and in vivo evaluation of two candidate antiviral compounds active against EV-A71, a viral capsid inhibitor (G197) and a novel host-targeting phosphatidylinositol 4-kinase III beta inhibitor (N373) which, especially when used in combination, can significantly improve the survival and pathology of infected mice.

Figure 1

Chemical structures of G197 and N373. (A) G197 is a structural chimera of a capsid inhibitor reported by Shia et al. (blue circle) and vapendavir (BTA-798) (red circle). (B) The general structure of N373 is as shown and R¹ represents the unique chemical group that is covered by patents owned by Curovir.

Figure 2

Effect of novel PI4KIIIβ and capsid inhibitors on the replication of EV-A71 in vitro. RD cells infected with EV-A71 was treated with each of the compounds at 5 different concentrations and 0 µM refers to control infected cells treated with 0.1% DMSO. Virus titres achieved at 12 h.p.i. was determined by viral plaque assays and represented as bars on each of the charts. Cell viability of drug-treated non-infected cells was assessed by alamarBLUE Cell Viability Reagent and expressed as a percentage compared to control DMSO-treated cells. Novel PI4KIIIβ inhibitors were: N314, N339, N354, N373, N377, R006, R036, R041, S002, S003, S007, S011 and S014. N373 was selected for further studies based on superior cell viability profile and inhibition of EV-A71 replication. G197 was found to be more effective in inhibiting EV-A71 replication than control capsid inhibitor BTA-798. Bars represent Log₁₀ virus titres (PFU/ml) and points represent cell viability across the different compound concentrations. Dashed lines indicate negative control virus titre (top line) and a 2 Log₁₀ unit reduction in virus titre (lower line). Statistical analysis for differences in virus titre due to drug treatment was performed with one-way ANOVA with Dunnett’s post-test: *(p < 0.05), **(p < 0.01), ***(p < 0.005).

Figure 3

Treatment with G197 and N373 resulted decreased viral protein translation and treatment with G197 before virus entry resulted in greater inhibition efficacy consistent with the behaviour of capsid binding inhibitors. (A) Post-treatment of infected cells with G197 and (B) N373 resulted in a decrease in VP2 and precursor VP0 expression. Full length blots available were included as Supplementary Information 1. (C) Time-of-addition assay with G197 and N373 at 1 µM and negative control 0.1% DMSO. Treatment of cells with G197 before 0 h result in an abolishment of virus replication and addition of after virus has entered the cells were only able to reduce virus titres by a maximum of 1 Log₁₀ unit and no inhibition was observed from 2 h.p.i. N373 treatment gradually lost efficacy with delayed treatment up to 2 h.p.i. to no inhibition at 8 h.p.i. (D) Treatment of cells with G197 and N373 during the 1 h virus inoculation reduced virus titres significantly and G197 was more effective than N373. Statistical analysis for differences in virus titre due to drug treatment was performed with one-way ANOVA with Dunnett’s post-test: ***(p < 0.005). (E) Thermal stability of EV-A71 was assessed by incubating virus suspensions at temperatures from 37 to 60 °C in the presence of 0.1% DMSO or 1 µM G197. The presence of G197 improved the thermal stability of virus particles at elevated temperatures.

Figure 4

Treatment with both N373 and G197 exhibited greater inhibition of EV-A71 replication compared to single compounds when cells were treated after virus inoculation and not before. RD cells treated with N373 and G197 at the indicated combinations and concentrations were infected with EV-A71 after compound removal. Combination treatment (N373 + G197) at both concentrations resulted in virus titres lower than either of the single compound treatments at both MOI = 1 (A) and MOI = 0.1 (B). (C) RD cells were pre-treated for 2 h with N373 and G197 at the indicated combinations and concentrations prior to infection at MOI = 1 and (D) MOI = 0.1. Combination treatment did not result in a significantly lower virus titre compared to treatment with G197 only and N373 slightly reduced virus titres at a lower MOI. Statistical analysis for differences in virus titre due to drug treatment was performed with one-way ANOVA with Dunnett’s post-test: *(p < 0.05), **(p < 0.01), ***(p < 0.005). Student’s t-test was performed to compare single compound treatments (G197 or N373) to combination treatment at the same concentration.

Figure 5

Treatment with both N373 and G197 improved survival and reduced muscle tissue pathology in EV-A71 infected mice. (A) Treatment regimen composed of a single dose 2 h prior to infection and 6 daily doses after infection. Animals were observed and sacrificed at 14 days post-infection for survival group and at 5 days post-infection for tissue harvesting group. (B) Survival of EV-A71 infected Balb/c neonates showed the greatest improvement when treated with both N373 and G197 followed by N373 only and a slight improvement with only G197 compared to control group (DMSO). (C) Haemotoxylin and eosin staining on harvested limb tissue of infected mice at 5 days post-infection showed breakdown of muscle tissue ranging from extensive in control group to negligible in combination treatment group (N373 + G197).

Figure 6

N373 and G197 inhibited virus replication of CV-A6, CV-A16 and E-7 to varying extents in vitro. RD cells infected at MOI = 1 with CV-A6, CV-A16 and E-7 were treated with compounds N373 and G197 at indicated combinations and concentrations. Virus supernatants harvested at experimental end-point were assessed by viral plaque assays to determine impact of compound treatments on virus replication. (A) Both N373 and G197 efficiently inhibited the replication of CV-A6 and (B) CV-A16. (C) N373 was able to efficiently inhibit E-7 replication in RD cells and G197 was only able to inhibit virus replication at the higher concentration of 1 µM. Statistical analysis for differences in virus titre due to drug treatment was performed with one-way ANOVA with Dunnett’s post-test: *(p < 0.05), **(p < 0.01), ***(p < 0.005).