In vitro and in vivo studies of a potent capsid-binding inhibitor of enterovirus 71.
Enterovirus 71 (EV-A71) is an important pathogen that can cause severe neurological symptoms and even death. Our aim was to identify potent anti-EV-A71 compounds and study their underlying mechanisms and in vivo activity.



We identified a potent imidazolidinone derivative (abbreviated to PR66) as an inhibitor of EV-A71 infection from the screening of compounds and subsequent structure-based modification. Time-course treatments and resistant virus selection of PR66 were employed to study the mode of mechanism of PR66. In vivo activity of PR66 was tested in the ICR strain of new-born mice challenged with EV-A71/4643/MP4.



PR66 could impede the uncoating process during viral infection via interaction with capsid protein VP1, as shown by a resistant virus selection assay. Using site-directed mutagenesis, we confirmed that a change from valine to phenylalanine in the 179th amino acid residue of the cDNA-derived resistant virus resulted in resistance to PR66. PR66 increased the virion stability of WT viruses, but not the PR66-resistant mutant, in a particle stability thermal release assay. We further showed that PR66 had excellent anti-EV-A71 activity in an in vivo mouse model of disease, with a dose-dependent increase in survival rate and in protection against virus-induced hind-limb paralysis following oral or intraperitoneal administration. This was associated with reductions of viral titres in brain and muscle tissues.



We demonstrated here for the first time that an imidazolidinone derivative (PR66) could protect against EV-A71-induced neurological symptoms in vivo by suppressing EV-A71 replication. This involved binding to and restricting viral uncoating.
Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan.
Jin-Yuan
JY
Ho
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Taiwan.
Jyh-Haur
JH
Chern
Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan.
Chung-Fan
CF
Hsieh
Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan.
Szu-Ting
ST
Liu
Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan.
Chien-Jou
CJ
Liu
Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan.
Ya-Sian
YS
Wang
Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan.
Ta-Wei
TW
Kuo
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Taiwan.
Sheng-Ju
SJ
Hsu
Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Taiwan.
Teng-Kuang
TK
Yeh
Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan.
Shin-Ru
SR
Shih
Research Center for Industry of Human Ecology and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Taoyuan, Taiwan Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
Pei-Wen
PW
Hsieh
Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan.
Cheng-Hsun
CH
Chiu
Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan Research Center for Industry of Human Ecology and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Taoyuan, Taiwan Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan jimtong@mail.cgu.edu.tw.
Jim-Tong
JT
Horng
D000818:Animals; D000998:Antiviral Agents / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D002213:Capsid / Q000187:drug effects*; D002460:Cell Line; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical; D029821:Enterovirus A, Human / Q000187:drug effects*; D004769:Enterovirus Infections / Q000188:drug therapy / Q000821:virology; D006801:Humans; D020128:Inhibitory Concentration 50; D008813:Mice, Inbred ICR; D016019:Survival Analysis
D000998:Antiviral Agents