The potential of leritrelvir repositioning for the treatment of coxsackievirus B4 and common enterovirus infections

Enteroviruses (EVs) pose major threats to human health, among which coxsackievirus B4 (CVB4) is particularly notable for its high lethality, increasing prevalence, and the absence of approved targeted therapeutics. Leritrelvir, an approved coronavirus disease 2019 (COVID-19) drug targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease (3CL^pro), was investigated for repurposing due to structural and functional homology between the EV 3C protease (3C^pro) and SARS-CoV-2 3CL^pro. Molecular docking analyses demonstrated that leritrelvir binds to the CVB4 3C^pro active site in a manner similar to that observed for SARS-CoV-2 3CL^pro. In vitro experiments showed that leritrelvir inhibited CVB4 replication in HeLa cells, with a half-maximal effective concentration (EC₅₀) of 12.64 nM. The half-maximal cytotoxic concentration (CC₅₀) of leritrelvir in HeLa cells was 572.7 μM, yielding a selectivity index (SI=CC₅₀/EC₅₀) of 4.53 × 10⁴. Among 3-day-old ICR mice infected with CVB4, leritrelvir suppressed viral replication in a dose-dependent manner. Administration of leritrelvir (12 mg/kg/day) prevented mortality, alleviated clinical symptoms and weight loss, and attenuated histopathological lesions in virus-targeted organs (e.g., brain, lungs, myocardium, and small intestine). Additionally, leritrelvir effectively suppressed CVB4-induced elevation of interleukin-6. Leritrelvir exhibited superior anti-CVB4 efficacy both in vitro and in vivo compared with the reference drug fluoxetine. Potent activity was observed against CVB4 clinical isolates and 3C^pro mutants, confirming broad-spectrum efficacy against CVB4 variants. In vitro and in silico analyses revealed that leritrelvir exerted robust antiviral effects against EVA71, CVA16, and human rhinovirus 2. These findings support the repurposing potential of leritrelvir against CVB4 and common EVs.