Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity

Abstract

Poliovirus (PV) and enterovirus 71 (EV71) cause severe neurological symptoms in their infections of the central nervous system. To identify compounds with anti-PV and anti-EV71 activities that would not allow the emergence of resistant mutants, we performed drug screening by utilizing a pharmacologically active compound library targeting cellular factors with PV and EV71 pseudoviruses that encapsidated luciferase-encoding replicons. We have found that metrifudil (N-[2-methylphenyl]methyl)-adenosine) (an A2 adenosine receptor agonist), N 6 -benzyladenosine (an A1 adenosine receptor agonist) and NF449 (4,49,40,409-[carbonylbis[imino-5,1,3-benzenetriyl bis(carbonyl-imino)]] tetrakis (benzene-1,3-disulfonic acid) octasodium salt) (a Gs-a inhibitor) have anti-EV71 activity, and that GW5074 (3-(3, 5-dibromo-4-hydroxybenzylidine-5-iodo-1,3-dihydro-indol-2-one)) (a Raf-1 inhibitor) has both anti-PV and anti-EV71 activities. EV71 mutants resistant to metrifudil, N 6 -benzyladenosine and NF449 were isolated after passages in the presence of these compounds, but mutants resistant to GW5074 were not isolated for both PV and EV71. The inhibitory effect of GW5074 was not observed in Sendai virus infection and the treatment did not induce the expression of OAS1 and STAT1 mRNA. Small interfering RNA treatment against putative cellular targets of GW5074, including Raf-1, B-Raf, Pim-1, -2, and -3, HIPK2, GAK, MST2 and ATF-3, did not consistently suppress PV replication. Moreover, downregulation of Raf-1 and B-Raf did not affect the sensitivity of RD cells to the inhibitory effect of GW5074. These results suggest that GW5074 has strong and selective inhibitory effect against the replication of PV and EV71 by inhibiting conserved targets in the infection independently of the interferon response.

Cellular kinase inhibitors that suppress enterovirus replication have a conserved target in viral protein 3A similar to that of enviroxime

“…Cytotoxicity of the compounds was evaluated by two methods: one was observation of cell viability at 7 h under the same conditions as used for the screening and the other was the determination of 50 % cytotoxic concentration (CC 50 ) of drugs by the measurement of ATP concentration as a marker of metabolically active cells, as described previously (Arita et al, 2008b). RD cells (1.4610 4 cells per well in 100 ml medium) were cultured at 37 uC in 96-well plates (Becton Dickinson), followed by addition of 100 ml compound solution in a range of concentrations (7.8-500 mM) for GW5074, AG1478, IGF-1R inhibitor II and Flt3 inhibitor II.…”

“…Virus RNA was reverse-transcribed by using the Reverse Transcription system (Promega) and then subjected to real-time PCR by using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) with primers (forward primer and reverse primer 2) and a probe (probe 1) (Nijhuis et al, 2002). The fluorescence emission of the probe was monitored and analysed by using the Applied Biosystems 7500 Fast Real-Time PCR system, as described previously (Arita et al, 2008b). …”

“…1a; Supplementary Table S1). The inhibitory effect of GW5074 has been observed in L20B (murine), HEp-2c and RD (human) cells and thus is not species-specific (Arita et al, 2008b). Therefore, we used RD cells for the present screenings because this cell line is susceptible to both PV and EV71.…”

“…Therefore, we used RD cells for the present screenings because this cell line is susceptible to both PV and EV71. Cells were inoculated with PV pseudoviruses that have a luciferase-encoding PV replicon genome encapsidated in a PV capsid, as described previously (Arita et al, 2006(Arita et al, , 2008b, and then the luciferase activity in the cells was measured at 7 h p.i. The mean luciferase activity in the mock-treated cells infected with PV pseudoviruses was 1.3610 6 relative light units (RLU), and that in GW5074-treated cells was 2.4610 4 RLU.…”

“…In our previous attempt to identify potent anti-enterovirus compounds that target cellular factors, we identified a Raf-1 inhibitor, GW5074, by screening pharmacologically active compounds (Arita et al, 2008b). However, inhibitors for the downstream signalling pathway of Raf-1 [MEK/extracellular signal-regulated kinase (ERK) signalling] did not affect PV replication.…”