In vitroandin vivostudies of a potent capsid-binding inhibitor of enterovirus 71

Abstract

Objectives: 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. Methods: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.Results: 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. Conclusions: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.

Inhibition of EV71 by curcumin in intestinal epithelial cells

“…The virus titer was decreased in curcumin-treated cells compared with untreated cells ( Fig 3B ). PR66, an imidazolidinone derivative, was used as a control in this experiment ( S2 Fig ) [ 15 ]. Interestingly, we found that in addition to EV71, curcumin also suppressed the replication of CVB3 and EV-D68 viruses in intestinal epithelial cells ( S3 Fig ).…”

Fexaramine as an entry blocker for feline caliciviruses

“…Virus entry inhibitors have been developed or reported for enveloped viruses including human immunodeficiency virus (HIV) (Haqqani and Tilton, 2013;Woollard and Kanmogne, 2015), influenza virus (Palese and Shaw, 2007), Measles virus (Plemper et al, 2005) and Dengue virus (Scaturro et al, 2014). Virus entry inhibitors have also been reported for non-enveloped viruses including picornaviruses including poliovirus (Thys et al, 2008), enterovirus (Buontempo et al, 1997;Ho et al, 2016;Ma et al, 2017;Torres-Torres et al, 2015) and rhinoviruses Kim et al, 2017;Tijsma et al, 2014). However, entry inhibitors of any calicivirus have not been reported and this is the first report on a small molecule entry inhibitor of feline calicivirus.…”

“…The process of cell entry by FCV and other non-enveloped virus is generally not well understood, but viral binding to the cellular receptor is believed to trigger a conformational change in the capsid leading to capsid disassembly and release of its contents for replication (Bhella et al, 2008;Johnson, 2010). Recently, virus entry has become one of the targets for therapeutic intervention, but only a few small molecule inhibitors targeting the virus capsid have been reported for non-enveloped viruses including T picornaviruses (Buontempo et al, 1997;De Palma et al, 2008;Ho et al, 2016;Kim et al, 2017;Ma et al, 2017;Romero, 2001;Thys et al, 2008;Tijsma et al, 2014;Torres-Torres et al, 2015).…”

Rosmarinic acid exhibits broad anti-enterovirus A71 activity by inhibiting the interaction between the five-fold axis of capsid VP1 and cognate sulfated receptors

“…The infected mice were monitored daily for body weight, survival, and disease score. The disease scores were set as follows to represent the progression of virus infection: score 1, slow movement; score 2, one hind-limb paralysis; score 3, both hindlimb paralysis; score 4, death [14]. Time-of-addition assay RD cells (5 × 10 5 cells/ well) were seeded in six-well plates for 24 h. The cells were then infected with various viruses at an multiplicity of infection (MOI) of 10 and incubated for 1 h at 4°C.…”

“…The acidic environment causes a pH-dependent conformational alteration of VP1 and dislodges the "pocket factor" from the hydrophobic pocket beneath the canyon region of the virions, permitting initiation of the uncoating process [2]. X-ray structures revealed that the WIN compounds, such as DBPR103 and PR66, replace the natural pocket factor within the hydrophobic pocket, stabilizing the viral particle and preventing virus uncoating [12][13][14]. Moreover, PSGL1, a pan-selectin ligand, is expressed on leukocytes, platelets, and endothelial cells.…”