Vascular endothelial growth factor receptor 2 as a potential host target for the inhibition of enterovirus replication

Because host kinases are key regulators of multiple signaling pathways in response to viral infections, we previously screened a kinase inhibitor library using rhabdomyosarcoma cells and human intestinal organoids in parallel to identify potent inhibitors against EV-A71 Infection. We found that Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor efficiently suppressed the EV-A71 replication and further revealed ROCK1 as a novel EV-A71 host factor. In this study, subsequent analysis found that a variety of vascular endothelial growth factor receptor (VEGFR) inhibitors also had potent Antiviral effects. Among the hits, Pazopanib, with a selectivity index as high as 254, which was even higher than that of Pirodavir, a potent broad-spectrum picornavirus inhibitor targeting viral capsid protein VP1, was selected for further analysis. We demonstrated that Pazopanib not only efficiently suppressed the replication of EV-A71 in a dose-dependent manner, but also exhibited broad-spectrum anti-enterovirus activity. Mechanistically, Pazopanib probably induces alterations in host cells, thereby impeding viral genome replication and transcription. Notably, VEGFR2/KDR/Flk-1 knockdown and overexpression suppressed and facilitated EV-A71 replication, respectively, indicating that VEGFR2/KDR/Flk-1 is a novel host dependency factor for EV-A71 replication. Transcriptome analysis further proved that VEGFR2/KDR/Flk-1 potentially plays a crucial role in combating EV-A71 Infection through the TSAd-Src-PI3K-Akt pathway. These findings expand the range of potential Antiviral candidates of anti-enterovirus therapeutics and suggest that VEGFR2/KDR/Flk-1 may be a key host factor involved in EV-A71 replication, making it a potential target for the development of anti-enterovirus therapeutics.

Importance: As the first clinical case was identified in the United States, EV-A71, a significant neurotropic Enterovirus, has been a common cause of hand, foot, and mouth disease (HFMD) in infants and young children. Developing an effective Antiviral agent for EV-A71 and other human enteroviruses is crucial, as these viral pathogens consistently cause outbreaks in humans. In this study, we demonstrated that multiple inhibitors against VEGFRs effectively reduced EV-A71 replication, with Pazopanib emerging as the top candidate. Furthermore, Pazopanib also attenuated the replication of other enteroviruses, including CVA10, CVB1, EV-D70, and HRV-A, displaying broad-spectrum anti-enterovirus activity. Given that Pazopanib targets various VEGFRs, we narrowed the focus to VEGFR2/KDR/Flk-1 using knockdown and overexpression experiments. Transcriptomic analysis suggests that Pazopanib's potential downstream targets involve the TSAd-Src-PI3K-Akt pathway. Our work may contribute to identifying targets for Antiviral inhibitors and advancing treatments for human Enterovirus infections.

EV-A71; VEGFR family; VEGFR2; host factor; kinase inhibitors.