(14,15) In the cases for known inhibitors of lipid kinases such as apilimod (PIKFyve) (16) and R-59-022 (diacyclglycerol kinase), (17) understanding the contributions of on-target mechanisms for blockage of viral entry could offer approaches to new antiviral agents. Agents targeting the required host factor TPC1 based on analogues of tetrandrine are under investigation, as are specific agents for the lysosomal NPC1 receptor. Of interest for clinical translation are broad-spectrum viral entry inhibitors with known pharmacological mechanisms of action toward host targets.
(10−13) This binding event, together with other environmental factors, is necessary for the eventual fusion of viral and vesicular membranes, releasing the viral contents into the cytoplasm where genome transcription and replication can initiate and produce new virus progeny. (8,9) The processed form of GP can interact with the lysosomal host cell receptor, Niemann Pick C-type 1 (NPC1). (3−7) EBOV viral particles use endosomes for delivery to lysosomes during which a reduction in pH activates cathepsins to cleave the viral glycoprotein (GP). EBOV cell entry involves engagement of the virus particle with receptors that include TIM-1, which triggers uptake via macropinocytosis. Viral entry into cells, where the virus first binds to cell surface receptors followed by endosomal trafficking before entering the cell cytoplasm, offers many important potential host targets. Filoviral pathogens are negative-stranded RNA viruses that encode only a few enzymes considered traditional targets for inhibition. Many viral pathogens share common steps in viral entry, suggesting the potential for broad-spectrum therapies. Overall, the compounds show improved selectivity and minimal cytotoxicity relative to classical endosomal acidification blocking agents. The results are consistent with the action of the diphyllin scaffold as a selective pH-dependent viral entry block in late endosomes. 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. Unlike general lysosomotrophic agents, the diphyllin derivatives showed no major disruptions of endocytic populations or morphology when examined with Rab5 and LAMP1 markers. The most potent effects were reversible exhibiting higher selectivity than bafilomycin or the parent diphyllin. Mechanistically, the diphyllin derivatives had no effect on uptake and colocalization of viral particles with endocytic marker LAMP1 but directly modulated endosomal pH. In cells challenged with Ebola virus, the diphyllin derivatives inhibit viral entry dependent upon structural variations to low nanomolar potencies.
CELLPROFILER MINIMIZING CONTRAST SERIES
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. EBOV cellular entry and infection involve uptake via macropinocytosis, navigation through the endocytic pathway, and pH-dependent escape into the cytoplasm. To date, there are no clinically effective small-molecule drugs for postexposure therapies to treat filoviral infections. Ebola virus (EBOV) is an aggressive filoviral pathogen that can induce severe hemorrhagic fever in humans with up to 90% fatality rate.
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