Fluoxazolevir inhibits hepatitis C virus infection in humanized chimeric mice by blocking viral membrane fusion

Fluoxazolevir is an aryloxazole-based entry inhibitor of hepatitis C virus (HCV). We show that fluoxazolevir inhibits fusion of HCV with hepatic cells by binding HCV envelope protein 1 (E1) to prevent fusion. 9 of 10 fluoxazolevir-resistance-associated substitutions are in the E1 protein and 4 are in a putative fusion peptide. Pharmacokinetic studies in mice, rats and dogs revealed that fluoxazolevir localizes to the liver. A four-week intraperitoneal regimen of fluoxazolevir in humanized chimeric mice infected with HCV genotype 1b, 2a or 3 resulted in a 2-log reduction in viremia, without evidence of drug resistance. In comparison, daclatasvir, an approved HCV drug, suppressed more than 3-log of viremia but is associated with emergence of resistance-associated substitutions in mice. Combination therapy using fluoxazolevir and daclatasvir cleared HCV genotypes 1b and 3 in mice. Fluoxazolevir combined with glecaprevir and pibrentasvir was also effective in clearing multidrug-resistant HCV replication in mice. Fluoxazolevir may be promising as the next generation of combination drug cocktails for HCV treatment.