Coste, M. | bioRxiv Science

bioRxiv2019

Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells

Zika virus (ZIKV), an emerging flavivirus which causes neurodevelopmental impairment to fetuses and has been linked to Guillain-Barre syndrome, continues to threaten global health due to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) have been employed to improve the bioavailability of ribonucleoside analogues. Here, we synthesized and tested a library of 13 ProTides against ZIKV in human neural stem cells. Strong activity was observed for 2'-C-methyluridine and 2'-C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking group. Conversion of the aryloxyl phosphoramidate ProTide group of 2'-C-methyluridine to a 2-(methylthio)ethyl phosphoramidate completely abolished antiviral activity of the compound. The aryloxyl phosphoramidate ProTide of 2'-C-methyluridine outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and protection from cytopathic effect, while the former compounds triphosphate active metabolite was better incorporated by purified ZIKV NS5 polymerase over time. Molecular superpositioning revealed different orientations of residues opposite the 2'-fluoro group of sofosbuvir. These findings suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue ProTides in a disease-relevant cell model.

microbiology↗

bioRxiv2018

Development and validation of a phenotypic high-content imaging assay for assessing the antiviral activity of small-molecule inhibitors targeting the Zika virus

Zika virus (ZIKV) has been linked to the development of microcephaly in newborns, as well as Guillain-Barre syndrome. There are currently no drugs available to treat infection, and accordingly there is an unmet medical need for discovery of new therapies. High-throughput drug screening efforts focusing on indirect readouts of cell viability are prone to a higher frequency of false positives in cases where the virus is viable in the cell but the cytopathic effect is reduced or delayed. Here, we describe a fast and label-free phenotypic high-content imaging assay used to detect cells affected by the viral-induced cytopathic effect (CPE) using automated imaging and analysis. Protection from CPE correlates with a decrease in viral antigen production as observed by immunofluorescence. We trained our assay using a collection of nucleoside analogues against ZIKV; the previously reported antiviral activities of 2-C-methylribonucleosides and ribavirin against the Zika virus in Vero cells were confirmed using our developed method. Profiling of a novel library of 24 natural product derivatives using our assay revealed compound 1 as an inhibitor of ZIKV-induced cytopathic effect; activity of the compound was confirmed in human fetal neural stem cells (NSCs). The described technique can be easily leveraged as a primary screening assay for profiling large compound libraries against ZIKV, and can be expanded to other ZIKV strains and other cell lines displaying morphological changes upon ZIKV infection.

microbiology↗