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Many viruses utilize endosomal pathways to gain entry to cells and propagate infection. Sensing of endosomal acidification is a trigger for release of many virus cores into the cell cytosol. Previous efforts with inhibitors of vacuolar-ATPase have been shown to block endosomal acidification and affect viral entry albeit with limited potential for therapeutic selectivity. Herein, four novel series of derivatives of the vacuolar-ATPase inhibitor diphyllin were synthesized to assess their potential for enhancing potency and anti-filoviral activity over cytotoxicity. Derivatives that suitably blocked cellular entry of Ebola pseudo-typed virus were further evaluated as inhibitors of endosomal acidification and isolated human Vacuolar-ATPase activity. Several compounds with significant increases in potency over diphyllin in these assays also separated from cytotoxic doses in human cell models by >100 fold. Finally, three derivatives were shown to be inhibitors of replication-competent Ebola viral entry into primary macrophages with comparable potencies and enhanced selectivity toward anti-viral activity.

Ebola virus (EBOV) is an aggressive filoviral pathogen that can induce severe hemorrhagic fever in humans with up to 90% fatality rate. To date, there are no clinically effective small-molecule drugs for postexposure therapies to treat filoviral infections. EBOV cellular entry and infection involve uptake via macropinocytosis, navigation through the endocytic pathway, and pH-dependent escape into the cytoplasm. We report the inhibition of EBOV cell entry via selective inhibition of vacuolar (V)-ATPase by a new series of phenol-substituted derivatives of the natural product scaffold diphyllin. In cells challenged with Ebola virus, the diphyllin derivatives inhibit viral entry dependent upon structural variations to low nanomolar potencies. Mechanistically, the diphyllin derivatives had no effect on uptake and colocalization of viral particles with endocytic marker LAMP1 but directly modulated endosomal pH. The most potent effects were reversible exhibiting higher selectivity than bafilomycin or the parent diphyllin. Unlike general lysosomotrophic agents, the diphyllin derivatives showed no major disruptions of endocytic populations or morphology when examined with Rab5 and LAMP1 markers. The dilated vacuole phenotype induced by apilimod treatment or in constitutively active Rab5 mutant Q79L-expressing cells was both blocked and reversed by the diphyllin derivatives. The results are consistent with the action of the diphyllin scaffold as a selective pH-dependent viral entry block in late endosomes. Overall, the compounds show improved selectivity and minimal cytotoxicity relative to classical endosomal acidification blocking agents.

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