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

Abstract

Zika virus (ZIKV), an emerging flavivirus which causes neurodevelopmental impairment 22 to fetuses and has been linked to Guillain-Barré syndrome, continues to threaten global health due 23 to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of 24 efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) 25 have been employed to improve the bioavailability of ribonucleoside analogues. Here, we 26 synthesized and tested a library of 13 ProTides against ZIKV in human neural stem cells. Strong 27 activity was observed for 2'-C-methyluridine and 2'-C-ethynyluridine ProTides with an aryloxyl 28 phosphoramidate masking group. Conversion of the aryloxyl phosphoramidate ProTide group of 29 2'-C-methyluridine to a 2-(methylthio)ethyl phosphoramidate completely abolished antiviral 30 activity of the compound. The aryloxyl phosphoramidate ProTide of 2'-C-methyluridine 31 outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and 32 protection from cytopathic effect, while the former compound's triphosphate active metabolite was 33 better incorporated by purified ZIKV NS5 polymerase over time. Molecular superpositioning 34 revealed different orientations of residues opposite the 2'-fluoro group of sofosbuvir. These findings 35 suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue 36 ProTides in a disease-relevant cell model. 37 38 cells, RNA-dependent RNA polymerase 39 40 1. Introduction 41The explosive spread of Zika virus (ZIKV) during the 2015-2016 epidemics in Latin America 42 attracted worldwide attention to this previously neglected disease. The lack of effective vaccines or 43 small molecules to prevent or treat this infection remains a cause for concern and emphasizes the 44 urgent need for new therapeutic options [1]. ZIKV, an emerging flavivirus infection, causes several 48 manifestations of the disease, and in rare cases, ZIKV infection has been linked to the 49 neuroinflammatory Guillain-Barré syndrome [2]. 50 Viral polymerases remain attractive drug targets for the development of selective antiviral 51 therapies [3-5]. Generally, clinically-approved inhibitors that target these proteins fall into two broad 52 classes [6,7]. The first class consists of nucleoside analogues that mimic the natural substrate of the 53 enzyme. Upon analogue incorporation by the virally-encoded polymerase, DNA or RNA synthesis 54 is abrogated by preventing further nucleotide incorporation (chain termination), thereby arresting 55 viral replication. The second class is known as non-nucleoside inhibitors, which bind allosterically 56 and arrest viral nucleic acid synthesis by distorting the polymerase active site geometry to interfere 57 with nucleotide binding or nucleotide incorporation. 58 A common prodrug strategy used for antiviral ribonucleoside analogues involves the chemical 59 synthesis of nucleoside analogue monophosphates with metabolically-removable masking groups 60 [8...

Live-cell RNA imaging with metabolically incorporated fluorescent nucleosides

“…While it does appear that ProTide-tC can diffuse across the plasma membrane, it is likely that unmasking is inefficient in HeLa cells, and further the compound appears to concentrate in cellular vesicles, precluding the use of the ProTide strategy for RNA metabolic labeling using tC and pyrroloC-derived nucleosides in this system. It is known from extensive work in medicinal chemistry that the effectiveness of phosphate group masking strategies is cell type-dependent (31,32), and accordingly such fluorescent nucleotides may have utility in other cell types.…”

Drugs for the Treatment of Zika Virus Infection

“…79−82 Recent in vitro studies of related nucleoside analogue ProTides have revealed that 2′-C-methyl and 2′-C-ethynyluridine aryoxyl phosphoramidate ProTides (6 and 7) show improved anti-ZIKV activity compared to sofosbuvir. 83 The authors note both a nucleobase and ProTide masking group bias in compound activity, the basis of which remains to be explored mechanistically. Sofosbuvir is FDA-approved for the treatment of HCV and is classified as a pregnancy category B drug, suggesting it may be an ideal candidate for acute dosing clinical trials for pregnant women.…”