A novel small-molecule compound disrupts influenza A virus PB2 cap-binding and inhibits viral replication.
The conserved residues 318-483 in the PB2 subunit of influenza A polymerase is an independently folded cap-binding domain (PB2cap) that exhibits a distinct binding mode from other host cap-binding proteins, which suggests that PB2cap might be an ideal drug target. This study aimed to identify a new class of anti-influenza inhibitors that specifically disrupts the interaction between PB2cap and host cap structures.



An innovative fluorescence polarization assay was established for primary screening, followed by cap-binding inhibitory activity, antiviral efficacy and cytotoxicity evaluations of the selected compounds. The best compound was characterized by multi-cycle virus growth assay, cross-protection test, synergism evaluation, mini-replicon assay, binding affinity analysis, docking simulation and mouse study.



Several PB2 cap-binding inhibitors were discovered. The compound 7-(4-hydroxy-2-oxo-2H-chromen-3-yl)-6H,7H,8H-chromeno[3',4':5,6]pyrano[3,2-c]chromene-6,8-dione, designated PB2-39, was identified as a potent inhibitor of replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 in vitro and H1N1, H5N1 and H7N9 in vivo. Combinational treatment with the influenza virus release inhibitor zanamivir and PB2-39 exerted a synergistic anti-influenza effect. Mechanistic experiments supported that PB2-39 suppressed viral polymerase activity. Docking and binding affinity analyses demonstrated that PB2-39 interacted with the PB2 cap-binding pocket, suggesting its role as a cap-binding competitor.



Our study provides new insights for the strategic development of novel cap-binding inhibitors of influenza A viruses.
Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
Shuofeng
S
Yuan
Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
Hin
H
Chu
Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
Ke
K
Zhang
Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
Jiahui
J
Ye
School of Biological Sciences, Faculty of Science, The University of Hong Kong, Hong Kong SAR, China.
Kailash
K
Singh
Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
Richard Y T
RY
Kao
School of Biological Sciences, Faculty of Science, The University of Hong Kong, Hong Kong SAR, China.
Billy K C
BK
Chow
Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
Jie
J
Zhou
Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China bzheng@hkucc.hku.hk.
Bo-Jian
BJ
Zheng
D000818:Animals; D000998:Antiviral Agents / Q000302:isolation & purification* / Q000494:pharmacology* / Q000633:toxicity; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004195:Disease Models, Animal; D004357:Drug Synergism; D005260:Female; D005454:Fluorescence Polarization; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects* / Q000502:physiology*; D008807:Mice, Inbred BALB C; D009976:Orthomyxoviridae Infections / Q000188:drug therapy; D039381:RNA Cap-Binding Proteins / Q000037:antagonists & inhibitors*; D016896:Treatment Outcome; D014764:Viral Proteins / Q000037:antagonists & inhibitors*; D014779:Virus Replication / Q000187:drug effects*
D000998:Antiviral Agents; C502893:PB2 protein, influenza virus; D039381:RNA Cap-Binding Proteins; D014764:Viral Proteins