Evaluation of Phenol-Substituted Diphyllin Derivatives as Selective Antagonists for Ebola Virus Entry

Abstract

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.

Bioactivities and Mechanisms of Action of Diphyllin and Its Derivatives: A Comprehensive Systematic Review

“…The mechanism research revealed that they modulated the endosomal pH. These results were consistent with the fact that diphyllin is a selective pH-dependent viral entry block in late endosomes [60]. In 2022, Michal Štefánik et al reported that diphyllin and diphyllinosides (Cleistanthin B 8) displayed broad-spectrum antiviral activities against multiple enveloped RNA and DNA viruses by blocking the replication of these viruses.…”

Enamel defects in Acp4R110C/R110C mice and human ACP4 mutations

“…S10 – S13 ). Lysosome-Associated Membrane Protein 1 (LAMP1) was used as a lysosomal marker 42 , RAB5 as an early endosome marker 43 , and clathrin (CLTC) as a marker for coated vesicles and coated pits involved in endocytosis 43 . In secretory stage ameloblasts, ACP4 immunostaining was restricted largely to the Tomes’ process, the specialized secretory organ comprising the distal membrane of secretory ameloblasts.…”