High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

Summary

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

Abstract

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

Introduction

RNA viruses are responsible for a large variety of human infections, and have a huge impact on populations worldwide both in terms of public health and economical cost. Efficient vaccines have been developed against several human RNA viruses, and are widely used as prophylactic treatments. However, there is still a critical lack of therapeutic drugs against RNA virus infections. Indeed, efficient vaccines are not available against major human pathogens such as dengue virus, Hepatitis C virus or human respiratory syncytial virus (hRSV). Besides, RNA viruses are responsible for a majority of emerging diseases, which have increased in frequency because of global exchange....

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Protocol

1. Preparation of 96-well Daughter Plates with Compounds

1. Move 96-well mother plates containing 10 mM stock solutions of chemical compounds in DMSO from -20 °C to room temperature. Dilute compounds 5x in DMSO to obtain intermediate 96-well dilution plates at 2 mM. Dilution is achieved by pipetting 5 μl into 20 μl of DMSO and mixing.
2. Pipette 1 μl from dilution plates into dry wells of white, bar-coded tissue culture 96-well plates. This first set of daughter plates (D1) will be used to evaluate the toxicity of compounds at a final concentration of 20 μM. Store daughter plates at -20 °C until use.
3. Di....

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Results

This screening pipeline relies first on the selection of compounds that inhibit MV replication and do not show any significant cellular toxicity (Figure 1, primary screen). It takes advantage of luciferase-based assays to determine cellular toxicity and the inhibition of viral replication. All pipetting steps and data acquisition can be performed in a high-throughput setting with a robotic platform.

Cellular toxicity of compounds is assessed using a luciferase-based viability .......

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Discussion

The screening pipeline described here aims at selecting compounds with a suitable profile as broad-spectrum inhibitors of RNA viruses. When a library of 10,000 compounds was screened with this protocol and aforementioned filtering criteria were applied, i.e. inhibition of MV replication superior to 75%, 40 compounds (0.4%) scored positive. Besides, about half of them showed some toxicity in the luciferase-based viability assay, and were disregarded for this reason. Finally, a dozen of hits readily available in l.......

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Materials

Name	Company	Catalog Number	Comments
Freedom EVO platform	TECAN		Robotic platform
96-well polystyrene cell culture microplates, white	Greiner Bio One	655083
CellTiter-Glo Luminescent Cell Viability Assay	Promega	G7570	Luciferase-based viability assay
Bright-Glo Luciferase Assay System	Promega	E2610	Reagent containing firefly luciferase substrate
Renilla-Glo luciferase Assay System	Promega	E2710	Reagent containing Renilla luciferase substrate
Britelite plus Reporter Gene Assay System	Perkin-Elmer	6016761	Reagent containing firefly luciferase substrate. Can be used as an alternative to Brigh-Glo reagent to determine luciferase activity.