A Novel MAPK Inhibitor With Potent Antiviral Activity Against Enterovirus 71

Enterovirus71(EV71)is the major causative agent of hand,foot and mouth disease,which threatens the health of infants and young children.In most cases,the diseases caused by EV71 are self-healing,but in infants and young children,severe infection with EV71 can lead to encephalitis,neurogenic pulmonary edema,aseptic meningitis and other fatal diseases.Currently,no drug has been approved for the severe lethal diseases caused by EV71.Viral infection and propagation require activation of the MAPK signal pathway,especially the p38-MAPK signal pathway,which is highly related with the replication of EV71.A growing number of MAPK inhibitors has been used in clinical trials against inflammatory diseases due to the excellent anti-inflammatory effect.Considering the inflammatory diseases caused by EV71,MAPK inhibitors might be effective for the treatment.This study aims to screen MAPK inhibitors of anti-EV71 activity,investigate the antiviral effect and explore the mechanism.Firstly,a MAPK compound library with 65 inhibitors was screened for their inhibitory effects on the replication of EV71 in vitro.RT-qPCR data for EV71 VP1 RNA copy number suggested that several inhibitors,such as AS602801,PD169316,BRAF inhibitor,GW5074 and TAK-632,could significantly decrease the copy number of EV71 VP1 RNA to below 10% of the control.PD169316,a specific p38 inhibitor,showed a striking inhibitory effect(decrease to 2%).SB203580 is a commonly used p38 inhibitor that has a similar structure to PD169316,and PD169316 showed stronger anti-viral activity than SB203580 in RD cells.The copies of EV71 VP1 RNA decreased to 11.13%,13.65% and 0.2% of the control with PD169316 treatment at 5 ?M,10 ?M and 20 ?M respectively at 24 hours post-infection(hpi),but the copies of EV71 VP1 RNA only decreased to 65%,70%and 47% of the control with SB203580 treatment at the respective concentrations.Similar results were exhibited in HeLa cells and MRC-5 cells.A better antiviral effect for PD169316 than for SB203580 also appeared on VP1 protein in RD cells and HeLa cells by Western blotting.The expression of EV71 VP1 was significantly decreased inHeLa cells after knockdown of p38 expression using p38 shRNA.20 ?M PD169316 did not show cytotoxicity on RD cells.Next we evaluated the antiviral efficacy of the inhibitors in vivo.EV71 GDV103 did not infect one-day-old Kunming suckling mice and three-week-old NOD-SCID mice,so another mouse-adapted EV71 MA was used in the evaluation of PD169316's antiviral effect.Suckling mice infected with EV71 MA exhibited four-limb paralysis or death.EV71 VP1 RNA copy detection from different organs showed the skeletal muscle is the predominant part of viral replication.The virulence of EV71 MA was enhanced by continuous passages in suckling mice,and the seven-day-old suckling mice model was chosen as the animal model for antiviral investigation against EV71 on several inhibitors.The suckling mice were intracranially infected with EV71 MA at 106 pfu,followed by intramuscular injection with the inhibitors(dissolved in DMSO)to achieve a dose of 1 mg/kg.Survival-curve exhibited that only PD169316-treated mice had a higher survival rate compared to the control(70% vs 40%).PD169316-treated mice succumbed to death apparently later than the control,although the difference in the survival rate between the two groups was not significant.The clinical scores in PD169316-treated group was better than the control.In the control group,the single limb paralysis appeared at 5 days post-challenge,and the symptoms continued to deteriorate,the four-limb paralysis or death appeared at 9 days post-challenge,whereas mice with PD169316 treatment presented paralysis of an average of two limbs at 9 days post-challenge.A significant difference in clinical manifestations was observed between the two groups at 11 days post-challenge.Except the survival and clinical scores,other antiviral effects of PD169316 were evaluated in vivo,viral RNA,viral titer and the antigen level of EV71 VP1 were significantly lowered by PD169316 in the skeletal muscle compared to the control.Histological examination by H&E staining showed that focal and patchy hemorrhages were found in the brain of the DMSO-treated mice,whereas no pathological change occurred in the PD169316-treated mice.In the intestine,submucosal inflammatory infiltration was identified in the DMSO-treated mice as well as the PD169316-treated mice,with improvement after PD169316 treatment.Skeletal muscle was widely damaged by EV71,displaying visible degeneration,edema,hemorrhage,inflammatory infiltration,partial necrotic myositis and diffuse lesions.With PD169316 treatment,the inflammatory infiltration and necrotic myositis werealleviated.The expression of proinflammatory cytokines in the serum was quantified by flow cytometry.At 5 days post-challenge,the levels of TNF-? and MCP-1 were obviously decreased in the group treated with PD169316 compared to the control.Together,PD169316 can inhibit the replication of EV71 and down-regulate the secretion of inflammatory cytokines in vivo.Next,PD169316's antiviral mechanism was explored.We found the replication of EV71 is indispensable for the activation of p38.The EV71 proteins,including four structural proteins(VP1,VP2,VP3,VP4)and seven non-structural proteins(2A,2B,2C,3A,3B,3C,3D)could not activate p38.The ?-propionolactone-inactivated EV71 virion also failed to induce the activation of p38.At the same time the replication of EV71 is indispensable for the caspase-3-midiated cell apoptosis,not EV71 proteins or the inactivated viron.PD169316 could inhibit cell apoptosis induced by EV71.Both of the intrinsic and extrinsic apoptosis intrigued by EV71 were attenuated with the treatment of PD169316 in a dose-dependent manner in RD cells and HeLa cells through Western blotting and flow cytometry analysis,and same results were gained by immunohistochemical staining with anti-cl-caspase-3 in tissues in mice.Next,STAT1 is an important molecular in the p38 activation and cell apoptosis.PD169316 could lower the level of STAT1's phosphorylation upon EV71 infection.Knockdown of STAT1 also significantly dampened the expressions of cl-caspase-3,cl-caspase-8,and cl-caspase-9.These findings illustrated that p38/p-STAT1Ser727 signal pathway was involved in EV71-induced cell apoptosis.Given that PD169316 could both decrease the replication of EV71 and cell apoptosis,we postulated that cell apoptosis could improve EV71's replication.We blocked the caspase-3 apoptosis pathway by DEVD-CHO,a specific caspase-3inhibitor,but the inhibition of cell apoptosis did not decrease viral replication.Accordingly,the antiviral activity of PD169316 was independent of caspase-3-midiated apoptosis.In conclusion,one p38-MAPK inhibitor,PD169316 was screened out for its anti-EV71 activity.The antiviral efficacy of PD169316 was confirmed in cell lines and an animal model.p38/STAT1Ser727 signal pathway was involved in the cell apoptosis induced by EV71,but viral replication was not dampened by the blockade of caspase-3-midiated apoptosis.This study thus offers a new opportunity for the development of a drug against EV71.