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

Abstract

Previously, we identified a cellular kinase inhibitor, GW5074, that inhibits poliovirus (PV) and enterovirus 71 replication strongly, although its target has remained unknown. To identify the target of GW5074, we searched for cellular kinase inhibitors that have anti-enterovirus activity similar or related to that of GW5074. With this aim, we performed screenings to identify cellular kinase inhibitors that could inhibit PV replication cooperatively with GW5074 or synthetically in the absence of GW5074. We identified MEK1/2 inhibitors (SL327 and U0126), an EGFR inhibitor (AG1478) and a phosphatidylinositol 3-kinase inhibitor (wortmannin) as compounds with a cooperative inhibitory effect with GW5074, and an Akt1/2 inhibitor (Akt inhibitor VIII) as a compound with a synthetic inhibitory effect with MEK1/2 inhibitors and AG1478. Individual treatment with the identified kinase inhibitors did not affect PV replication significantly, but combined treatment with MEK1/2 inhibitor, AG1478 and Akt1/2 inhibitor suppressed the replication synthetically. The effect of AG1478 in this synthetic inhibition was compensated by other receptor tyrosine kinase inhibitors (IGF-1R inhibitor II and Flt3 inhibitor II). We isolated mutants resistant to Flt3 inhibitor II and GW5074 and found that these mutants had crossresistance to each treatment. These mutants had a common mutation in viral protein 3A that results in an amino acid change at position 70 (Ala to Thr), a mutation that was previously identified in mutants resistant to a potent anti-enterovirus compound, enviroxime. These results suggest that cellular kinase inhibitors and enviroxime have a conserved target in viral protein 3A to suppress enterovirus replication. INTRODUCTIONThe genus Enterovirus consists of at least ten species of non-enveloped viruses with a single-stranded, positivesense genomic RNA that belong to the family Picornaviridae. Enterovirus infection is mostly asymptomatic, but sometimes causes severe neurological symptoms exemplified by poliomyelitis. In infection by poliovirus (PV), which is the causative agent of poliomyelitis and belongs to the species Human enterovirus C, motor neurons are the major target for neurovirulence (Bodian, 1949). Enterovirus 71 (EV71), another neurotropic enterovirus belonging to the species Human enterovirus A, is a causative agent of hand, foot and mouth disease and herpangina, but sometimes causes severe neurological diseases, such as brainstem encephalitis and poliomyelitis-like paralysis (Chumakov et al., 1979;McMinn, 2002;Wang et al., 2003). EV71 causes fatal pulmonary oedema and pulmonary haemorrhage in young children by destruction of the vasomotor and respiratory centres in the brainstem Ho et al., 1999;Huang et al., 1999;Komatsu et al., 1999;Lum et al., 1998;Wang et al., 1999). For PV, live-attenuated and inactivated vaccines have been established and are currently being used for global eradication of poliomyelitis (Sabin, 1965;Salk et al., 1954). However, there is no effective therapeutic means for vaccine-associated...

Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

“…Consistent with our speculation, the mutants could replicate significantly better than wt CVB3 in the presence of PIK93. While this work was in progress, a similar observation was made with the enviroxime-and GW5074-resistant PV A70T mutant [7,11], which was also found to provide protection against PIK93 [32].…”

“…GW5074 has previously also been reported to inhibit PV and EV71 replication [29]. Interestingly, PV mutants resistant against GW5074 contained the same point mutation (i.e., mutation A70T in the hydrophobic domain of 3A) as mutants resistant to enviroxime [11]. This finding prompted us to test whether our previously identified enviroxime-resistance mutations in CVB3 3A (i.e., mutations V45A and H57Y in the cytosolic domain of 3A) also provided cross-resistance to GW5074 [12].…”

“…Enviroxime-resistant PV, HRV, and CVB3 were all found to carry mutations in 3A, but the resistance mechanism has remained elusive [7,8,10]. Remarkably, PV mutants that are resistant to GW5074, a kinase inhibitor that inhibits PV RNA replication, were found to contain the same amino acid alteration as mutants resistant to enviroxime [11]. Intriguingly, we recently identified TTP-8307 as another inhibitor of enterovirus replication and found that TTP-8307-resistant CVB3 contained other mutations in 3A (i.e., V45A and H57Y) that also conferred resistance to enviroxime [12].…”

Lipid Involvement in Viral Infections: Present and Future Perspectives for the Design of Antiviral Strategies

“…Related reports have also uncovered that this enzyme is the cellular target of known antiviral compounds against enteroviruses [109]. This is so for some enviroxime-like compounds − T-00127-HEV1 and GW5074 [93] − that integrate a group of antivirals that inhibits enterovirus replication, for which mutations conferring drug resistance mapped to the same region of the enteroviral protein 3A [109,[203][204][205]. The recruitment of PI4KIII to viral replication complexes requires the participation of cellular partners like the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), as described for AiV [108,122].…”

Poliovirus Studies during the Endgame of the Polio Eradication Program

“…Host-targeting antivirals have the advantage of limiting emergence of resistance mutants (81,82), which arise from modulation of the viral/host proteins interaction (for brefeldin A) or from changing the ratio of viral protein expression (for PI4KB/OSBP inhibitors) (83,84), and its broad antiviral spectrum on picornavirus. However, the utility of a promising candidate, PI4KB inhibitor (27,28,(85)(86)(87)(88)(89)(90)(91), is currently limited because of its in vivo toxicity (92,93). In addition to chemical compounds, monoclonal antibodies that can neutralize PVs of different serotypes might be useful as anti-PV therapeutics (94).…”