Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects
Elena Bekerman, Gregory Neveu, Ana Shulla, Jennifer Brannan, Szu-Yuan Pu, Stanley Wang, Fei Xiao, Rina Barouch-Bentov, Russell R. Bakken, Roberto Mateo, Jennifer Govero, Claude M. Nagamine, Michael S. Diamond, Steven De Jonghe, Piet Herdewijn, John M. Dye, Glenn Randall, Shirit Einav
Elena Bekerman, Gregory Neveu, Ana Shulla, Jennifer Brannan, Szu-Yuan Pu, Stanley Wang, Fei Xiao, Rina Barouch-Bentov, Russell R. Bakken, Roberto Mateo, Jennifer Govero, Claude M. Nagamine, Michael S. Diamond, Steven De Jonghe, Piet Herdewijn, John M. Dye, Glenn Randall, Shirit Einav
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Research Article Virology

Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects

Abstract

Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses.

Authors

Elena Bekerman, Gregory Neveu, Ana Shulla, Jennifer Brannan, Szu-Yuan Pu, Stanley Wang, Fei Xiao, Rina Barouch-Bentov, Russell R. Bakken, Roberto Mateo, Jennifer Govero, Claude M. Nagamine, Michael S. Diamond, Steven De Jonghe, Piet Herdewijn, John M. Dye, Glenn Randall, Shirit Einav

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Figure 6

Mechanisms underlying the antiviral effect of sunitinib and erlotinib in vitro and in vivo.

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Mechanisms underlying the antiviral effect of sunitinib and erlotinib in...
(A–C) Huh7 cells were treated with the inhibitors and monitored for DENV entry (A) at 6 hours after infection, DENV RNA replication (B) after induction of replication of DNA-launched DENV replicon, and infectious virus production (C) at 48 hours after electroporation with DENV RNA. SDM25N is an inhibitor of DENV RNA replication. (D) Effect of 1-hour treatment with erlotinib (E) and/or sunitinib (SM) on phosphorylation of AP2 in DENV-infected Huh7 cells measured by Western blotting. Arrow indicates approximately 50 kDa. The ratio of phospho-AP2 (pAP2) to total AP2 was quantified. (E) Level of AP2 and actin expression measured by Western blot following lentiviral transduction with control or AP2-expressing constructs. (F) Rescue of DENV infection in the presence of inhibitors upon overexpression of WT or T156A AP2 versus vector control measured by luciferase assays 48 hours after infection. Micromolar concentration of each inhibitor is noted on the x axis. (G) Effect of 3-hour i.p. treatment with erlotinib (E) and/or sunitinib (SM) on phosphorylation of AP2 in liver tissue of AG-B6 mice measured by Western blotting and quantified as the ratio of pAP2 to total AP2. (H) DENV infection relative to NT control following siRNA-mediated knockdown of kinases targeted by sunitinib and erlotinib measured by luciferase assays at 48 hours and normalized to cell viability. Data in A, C, and I are pooled from 2 independent experiments with 4–8 replicates each. Data in the other panels are representative of 2 or more independent experiments. B and F have at least 5 replicates each. ***P < 0.001 relative to DMSO by 2-way ANOVA followed by Dunnett’s multiple comparisons test (B) or relative to vector control by 1-way ANOVA followed by Dunnett’s multiple comparisons test (F).