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

Abstract

Zika virus (ZIKV) has been linked to the development of microcephaly in newborns, as well as Guillain-Barré 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.

Anti‐Zika Virus Activity of Plant Extracts Containing Polyphenols and Triterpenes on Vero CCL‐81 and Human Neuroblastoma SH‐SY5Y Cells

“…There are few reports on the activity of natural products against ZIKV-infected SH-SY5Y cells, comprising isoquercitrin (assayed at 100 μM), [23] an omega-3-polyunsaturated fatty acid (assayed at 12.5 μM), [32] and the bacterial metabolite bafilomycin A1 (assayed at 10 nM). [33] Additionally, some natural products have been reported to exert anti-ZIKV activity in other neuronal cells, including chloroquine, assayed at 50 μM and 12.5 μM, respectively in human fetal neuronal stem cells [34] and in mouse neurospheres, [35] in addition to betulinic acid (assayed at 50 μM) in human neural progenitor cells. [36] The extracts of M. rigida, E. grandiflorus, M. ilicifolia (dichloromethane), and T. phaeocarpa had their 50 % cytotoxic concentration (CC 50 ) determined in Vero CCL-81 and SH-SY5Y cells by the MTT assay.…”

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

“…(3R,4R,5R)-5-(benzoyloxy)methyl)-3-ethynyltetrahydrofuran-2,3,4-triyl tribenzoate 1 was synthesized by following a previously reported procedure [27]. Synthetic details for aryloxy phosphoramidate ProTides other than for 2′- C -ethynyluridine and 2′- C -ethenyluridine were published previously by our group [24]. All reactions were carried out in either an oven-dried round bottom flask or Schlenk tube under a nitrogen atmosphere, using commercially available anhydrous solvents, and monitored by thin-layer chromatography, with detection by UV light.…”

“…Further, sofosbuvir is the only inhibitor tested against ZIKV thus far in ProTide form. In addition, recent work by our group and others has suggested that sofosbuvir and ProTides in general have differential activity depending on the cell line used, which may be linked to the cell-specific metabolism of ProTides[17,24]. In this work, we chemically synthesized a library of 13 ProTides and tested them for activity against ZIKV in human neural stem cells, a disease-specific cell model of infection.…”

Drugs for the Treatment of Zika Virus Infection

“…The anti-HCV drug 9 (ribavirin) has been reported to have potent anti-ZIKV activity in vitro and in a STAT-1 deficient mouse model of ZIKV infection. 74,86 While the mechanism of action of ribavirin remains under some debate, it is believed to act through a combination of RNA-synthesis abrogation upon incorporation by the RdpRp and through the induction of lethal mutagenesis via mismatch incorporation opposite the incorporated inhibitor in the template strand.…”