title: Discovery of itraconazole with broad-spectrum in vitro antienterovirus activity that targets nonstructural protein 3A.
abstract: There is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections, which remain a substantial threat to public health. To discover inhibitors that can be immediately repurposed for treatment of enterovirus infections, we developed a high-throughput screening assay that measures the cytopathic effect induced by enterovirus 71 (EV71) to screen an FDA-approved drug library. Itraconazole (ITZ), a triazole antifungal agent, was identified as an effective inhibitor of EV71 replication in the low-micromolar range (50% effective concentrations [EC50s], 1.15 μM). Besides EV71, the compound also inhibited other enteroviruses, including coxsackievirus A16, coxsackievirus B3, poliovirus 1, and enterovirus 68. Study of the mechanism of action by time-of-addition assay and transient-replicon assay revealed that ITZ targeted a step involved in RNA replication or polyprotein processing. We found that the mutations (G5213U and U5286C) conferring the resistance to the compound were in nonstructural protein 3A, and we confirmed the target amino acid substitutions (3A V51L and 3A V75A) using a reverse genetic approach. Interestingly, posaconazole, a new oral azole with a molecular structure similar to that of ITZ, also exhibited anti-EV71 activity. Moreover, ITZ-resistant viruses do not exhibit cross-resistance to posaconazole or the enviroxime-like compound GW5074, which also targets the 3A region, indicating that they may target a specific site(s) in viral genome. Although the protective activity of ITZ or posaconazole (alone or in combination with other antivirals) remains to be assessed in animal models, our findings may represent an opportunity to develop therapeutic interventions for enterovirus infection.
authors.affiliation: Institute of Molecular Ecology and Evolution, SKLEC & IECR, East China Normal University, Shanghai, China Unit of Anti-Infective Research, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Gao
authors.affiliation: Unit of Anti-Infective Research, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Shilin
authors.initials: S
authors.lastname: Yuan
authors.affiliation: Unit of Anti-Infective Research, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Institute of Molecular Ecology and Evolution, SKLEC & IECR, East China Normal University, Shanghai, China Unit of Anti-Infective Research, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Unit of Anti-Infective Research, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Yizhuo
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Institute of Molecular Ecology and Evolution, SKLEC & IECR, East China Normal University, Shanghai, China.
authors.forename: Guimei
authors.initials: G
authors.lastname: He
authors.affiliation: Institute of Molecular Ecology and Evolution, SKLEC & IECR, East China Normal University, Shanghai, China syzhang@bio.ecnu.edu.cn raltmeyer@ips.ac.cn gangzou@ips.ac.cn.
authors.forename: Shuyi
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Unit of Anti-Infective Research, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China syzhang@bio.ecnu.edu.cn raltmeyer@ips.ac.cn gangzou@ips.ac.cn.
authors.forename: Ralf
authors.initials: R
authors.lastname: Altmeyer
authors.affiliation: Unit of Anti-Infective Research, Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China syzhang@bio.ecnu.edu.cn raltmeyer@ips.ac.cn gangzou@ips.ac.cn.
authors.forename: Gang
authors.initials: G
authors.lastname: Zou
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D024882:Drug Resistance, Viral / Q000235:genetics; D004770:Enterovirus / Q000187:drug effects / Q000472:pathogenicity; D006801:Humans; D017964:Itraconazole / Q000494:pharmacology*; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D017361:Viral Nonstructural Proteins; D017964:Itraconazole