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Development and Validation of a Phenotypic High-Content Imaging Assay for Assessing the Antiviral Activity of Small-Molecule Inhibitors Targeting 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 ZIKV infection, and accordingly, there is an unmet medical need for the 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 (CPE) is reduced or delayed. Here, we describe a fast and label-free phenotypic high-content imaging assay to detect cells affected by the virus-induced CPE using automated imaging and analysis. Protection from the CPE correlates with a decrease in viral antigen production, as observed by immunofluorescence. We trained our assay using a collection of nucleoside analogues with activity 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. To validate the ability of our assay to reveal new anti-ZIKV compounds, we profiled a novel library of 24 natural product derivatives and found compound 1 to be an inhibitor of the ZIKV-induced cytopathic effect; the 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 of the activities of large compound libraries against ZIKV and can be expanded to other ZIKV strains and other cell lines displaying morphological changes upon ZIKV infection.

SUBMITTER: Bernatchez JA

PROVIDER: S-EPMC6153842 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Development and Validation of a Phenotypic High-Content Imaging Assay for Assessing the Antiviral Activity of Small-Molecule Inhibitors Targeting Zika Virus.

Bernatchez Jean A JA Yang Zunhua Z Coste Michael M Li Jerry J Beck Sungjun S Liu Yan Y Clark Alex E AE Zhu Zhe Z Luna Lucas A LA Sohl Christal D CD Purse Byron W BW Li Rongshi R Siqueira-Neto Jair L JL

Antimicrobial agents and chemotherapy 20180924 10

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 ZIKV infection, and accordingly, there is an unmet medical need for the 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 (CPE) is reduced or del ...[more]

PMID: 30061280

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Project description:The 26S proteasome is the principal protease for regulated intracellular proteolysis. This multi-subunit complex is also pivotal for clearance of harmful proteins that are produced throughout the lifetime of eukaryotes. Recent structural and kinetic studies have revealed a multitude of conformational states of the proteasome in substrate-free and substrate-engaged forms. These conformational transitions demonstrate that proteasome is a highly dynamic machinery during substrate processing that can be also controlled by a number of proteasome-associated factors. Essentially, three distinct family of deubiquitinases-USP14, RPN11, and UCH37-are associated with the 19S regulatory particle of human proteasome. USP14 and UCH37 are capable of editing ubiquitin conjugates during the process of their dynamic engagement into the proteasome prior to the catalytic commitment. In contrast, RPN11-mediated deubiquitination is directly coupled to substrate degradation by sensing the proteasome's conformational switch into the commitment steps. Therefore, proteasome-bound deubiquitinases are likely to tailor the degradation events in accordance with substrate processing steps and for dynamic proteolysis outcomes. Recent chemical screening efforts have yielded highly selective small-molecule inhibitors for targeting proteasomal deubiquitinases, such as USP14 and RPN11. USP14 inhibitors, IU1 and its progeny, were found to promote the degradation of a subset of substrates probably by overriding USP14-imposed checkpoint on the proteasome. On the other hand, capzimin, a RPN11 inhibitor, stabilized the proteasome substrates and showed the anti-proliferative effects on cancer cells. It is highly conceivable that these specific inhibitors will aid to dissect the role of each deubiquitinase on the proteasome. Moreover, customized targeting of proteasome-associated deubiquitinases may also provide versatile therapeutic strategies for induced or repressed protein degradation depending on proteolytic demand and cellular context.

Dataset Information

Development and Validation of a Phenotypic High-Content Imaging Assay for Assessing the Antiviral Activity of Small-Molecule Inhibitors Targeting Zika Virus.

Publications

Development and Validation of a Phenotypic High-Content Imaging Assay for Assessing the Antiviral Activity of Small-Molecule Inhibitors Targeting Zika Virus.

Similar Datasets

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