Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Abstract

Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primertemplate pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn 2ϩ is required for enzymatic activity, while Mg 2ϩ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5=-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2=-C-methyl-and 2=-C-ethynyl-substituted analog 5=-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

The A–Z of Zika drug discovery

“…The analogs showing both abilities were 2 0 -F-2-C-ME-UTP (IC 50 = 90.76 mM), 2 0 -C-ME-UTP (IC 50 = 5.78 mM) and 2 0 -C-ethynyl_UTP (IC 50 = 0.46 mM) ( Fig. 3e) [99]. ATP analogs were tested in NS5 polymerase in another study, and the compounds that led to the strongest enzyme inhibition were the 2 0 -Cmethylated ATPs [100].…”

“…(c) NS3 helicase inhibitor: suramin (EC 50 = 0.42 mM), which was tested only in a cell-based assay in ZIKV [75]. (d) NS5 polymerase inhibitors: sofosbuvir (IC 50 = 7.3 mM) [105], 2-C-ethynyl-UTP (IC 50 = 0.46 mM) [99] and DMB213 (IC 50 = 5.2 mM) [105]. (e) NS5 methyltransferase inhibitor: sinefungin (IC 50 = 1.18 mM) [90,91].…”

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

“…Some of the selected compounds, e.g. the 3′-deoxy and the 3′- 0 -methyl, are obligate chain terminators, but phosphorylation of this class of compounds in cells is often inefficient [18,19].…”

“…Single nucleotide incororation assays were modeled from previous work [19]. Briefly, ZIKV RdRP (1 μM) and P/T substrate (200 nM) were pre-incubated at 37°C for 20 min.…”

Zika Virus: What Have We Learnt Since the Start of the Recent Epidemic?

“…In addition, the expression of NS4A has been also related to activation of the cellular stress pathway involving Tor1 and type 2A phosphatase activator Tip41 (Li G. et al, 2017). NS5 is the viral RNA-dependent RNA polymerase that is in charge of genome replication constituting a major target for antiviral design (Lu et al, 2017;Xu et al, 2017). Furthermore, the analysis of the structure of the methyltransferase domain of NS5, which is responsible for capping the 5 end of the viral genomic RNA, also provides new opportunities for the design of antiviral compounds (Coloma et al, 2016;Stephen et al, 2016;Coutard et al, 2017;Zhou et al, 2017).…”