The Pyrimidine Analog FNC Potently Inhibits the Replication of Multiple Enteroviruses

Human enteroviruses (EVs), including coxsackieviruses, the numbered enteroviruses, and echoviruses, cause a wide range of diseases, such as hand, foot, and mouth disease (HFMD), encephalitis, myocarditis, acute flaccid myelitis (AFM), pneumonia, and bronchiolitis. Therefore, broad-spectrum anti-EV drugs are urgently needed to treat EV infection. Here, we demonstrate that FNC (2'-deoxy-2'-β-fluoro-4'-azidocytidine), a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs, including enterovirus 71 (EV71), coxsackievirus A16 (CA16), CA6, EVD68, and coxsackievirus B3 (CVB3), at the nanomolar level. The antiviral mechanism of FNC involves mainly positive- and negative-strand RNA synthesis inhibition by targeting and competitively inhibiting the activity of EV71 viral RNA-dependent RNA polymerase (3D^pol), as demonstrated through quantitative real-time reverse transcription-PCR (RT-qPCR), in vitro 3D^pol activity, and isothermal titration calorimetry (ITC) experiments. We further demonstrated that FNC treatment every 2 days with 1 mg/kg of body weight in EV71 and CA16 infection neonatal mouse models successfully protected mice from lethal challenge with EV71 and CA16 viruses and reduced the viral load in various tissues. These findings provide important information for the clinical development of FNC as a broad-spectrum inhibitor of human EV pathogens.IMPORTANCE Human enterovirus (EV) pathogens cause various contagious diseases such as hand, foot, and mouth disease, encephalitis, myocarditis, acute flaccid myelitis, pneumonia, and bronchiolitis, which have become serious health threats. However, except for the EV71 vaccine on the market, there are no effective strategies to prevent and treat other EV pathogen infections. Therefore, broad-spectrum anti-EV drugs are urgently needed. In this study, we demonstrated that FNC, a small nucleoside analog inhibitor that has been demonstrated to be a potent inhibitor of HIV and entered into a clinical phase II trial in China, potently inhibits the viral replication of a multitude of EVs at the nanomolar level. Further investigation revealed that FNC inhibits positive- and negative-strand RNA synthesis of EVs by interacting and interfering with the activity of EV71 viral RNA-dependent RNA polymerase (3D^pol). Our findings demonstrate for the first time that FNC is an effective broad-spectrum inhibitor for human EV pathogens.