Voxtalisib inhibits enterovirus 71 replication by downregulating host RAN and restoring IFN-STAT signaling.

Introduction: Hand, foot, and mouth disease (HFMD) is a common enterovirus-induced illness primarily affecting children under 5 years of age. Enterovirus 71 (EV71) is a major causative agent associated with severe HFMD that can lead to fatal neurological complications. Despite available vaccines, no antiviral therapy with a clearly defined molecular mechanism has received approval. While most antiviral agents target viral components, host-targeting antiviral approaches remain underexplored. We first identified voxtalisib, a dual PI3K/mTOR inhibitor, through compound screening as a potential inhibitor of EV71 replication; however, the underlying mechanism remains unclear.

Objectives: To identify novel host-targeted antiviral candidates and to investigate the antiviral mechanism of voxtalisib against EV71 and other enteroviruses.

Methods: Voxtalisib was first identified as a potent anti-EV71 compound through in vitro phenotypic screening. Follow-up experiments in rhabdomyosarcoma (RD) cells were used to assess the effect of voxtalisib on EV71 replication, and proteomic analysis identified its molecular targets. Voxtalisib's antiviral activity was also assessed against Coxsackie B viruses (CVB3, CVB4-5, and CVB4-7) and Echovirus 11 (Echo11). In vivo, suckling ICR mice were treated with voxtalisib. Survival rates, viral loads, and histopathological changes were subsequently evaluated.

Results: Proteomics analysis identified Ras-related nuclear protein (RAN) as a key host factor in EV71 replication. EV71 infection significantly upregulated RAN, while voxtalisib suppressed RAN. Mechanistically, RAN regulates nuclear-cytoplasmic transport of phosphorylated STAT1/2 (p-STAT1/2), affecting the interferon (IFN)-mediated antiviral response. Consequently, downregulating RAN expression enhanced the nuclear retention of p-STAT1/2 and upregulated interferon-stimulated genes expression, ultimately reducing EV71 replication. In vivo, voxtalisib improved survival, decreased viral loads, and alleviated organ damage in EV71-infected ICR suckling mice. Similar RAN-dependent p-STAT nuclear retention and antiviral effects were also observed against CVB and Echo11, confirming voxtalisib's broad-spectrum antiviral potential.

Conclusion: RAN is a novel antiviral host target that indirectly mediates EV71 replication by regulating the nuclear-cytoplasmic transport of p-STAT1/2. Voxtalisib effectively restores IFN-STAT signaling by modulating RAN, offering a promising host-directed antiviral strategy against enteroviruses.