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 exchanges and human impact on ecological systems. Against this threat that represent RNA viruses, our therapeutic arsenal is extremely limited and relatively inefficient^1-3. Current therapies are essentially based on recombinant type I interferons (IFN-α/β) to stimulate innate immunity, or the administration of ribavirin. Although the mode of action of this ribonucleoside analog is controversial and probably relies on various mechanisms, the inhibition of cellular IMPDH (inosine monophosphate dehydrogenase), which depletes intracellular GTP pools, is clearly essential⁴. Ribavirin, in combination with pegylated IFN-α, is the main treatment against Hepatitis C virus. However, IFN-α/β and ribavirin treatments are of relatively poor efficacy in vivo against most RNA viruses as they efficiently blunt IFN-α/β signaling through expression of virulence factors⁵ and often escape ribavirin³. This added to the fact that ribavirin treatment is raising important toxicity issues, although it was recently approved against severe hRSV disease with controversial benefits⁶. More recently, some virus-specific treatments have been marketed, in particular against influenza virus with the development of neuraminidase inhibitors³. However, the large diversity and permanent emergence of RNA viruses precludes the development of specific treatments against each one of them in a relatively close future. Altogether, this stresses the need for efficient strategies to identify and develop potent antiviral molecules in the near future.

It is trivial to say that a broad-spectrum inhibitor active against a large panel of RNA viruses would solve this problem. Although such a molecule is still a virologist's dream, our better understanding of cellular defense mechanisms and innate immune system suggest that some possibilities exist^7,8. Several academic and industrial laboratories are now seeking molecules that stimulate specific facets of cellular defense mechanisms or metabolic pathways to blunt viral replication. Although such compounds will probably show significant side effects, treatments against acute viral infections will be administered for a relatively short time, making them acceptable despite some potential toxicity on the long term. Various strategies have been developed to identify such broad-spectrum antiviral molecules. Some research programs aim at finding molecules that target specific defense or metabolic pathways. This includes, for example, pathogen recognition receptors to elicit antiviral gene expression⁹ and activate antiviral factors such as RNaseL¹⁰, autophagy machinery to promote virus degradation¹¹, nucleoside synthesis pathways^12,13, or apoptotic cascades to precipitate death of virus-infected cells¹⁴. Other groups have developed phenotypic screens that are not target-based^13,15-17. In that case, antiviral molecules are simply identified by their capacity to block viral replication in a given cellular system. The general assumption is that a compound inhibiting 2-3 unrelated RNA viruses would have a suitable profile for a broad-spectrum antiviral molecule. The mode of action of hit compounds selected with such an empirical approach is only determined in a second time and eventually, may lead to the identification of novel cellular targets for antivirals. Interestingly, a retrospective analysis of new drugs approved by the US Food and Drug Administration between 1999 and 2008 has shown that in general, such phenotypic screenings tend to perform better than target-based approaches to discover first-in-class small-molecule drugs¹⁸.

Viral replication in high-throughput cell-based assays is usually determined from virus cytopathic effects. Cells are infected and cultured in 96- or 384-well plates in the presence of tested compounds. After few days, cellular layers are fixed and stained with dyes such as crystal violet. Finally, absorbance is determined with a plate reader and compounds inhibiting viral replication are identified by their capacity to preserve cellular layers from virus-induced cytopathic effect. Alternatively, virally-mediated cytopathic effects are assessed using standard viability assays such as MTS reduction. Such assays are highly tractable and cost-effective, but suffer from three major limitations. First, they require a virus-cell combination where viral replication is cytopathic in only few days but this is not always possible, thus calling for alternative approaches¹⁹. Second, they are poorly quantitative since they are based on an indirect measure of viral replication. Finally, toxic compounds can be scored as positive hits, and therefore must be eliminated with a counter screen measuring cellular viability. To overcome some of these hurdles, recombinant viruses or replicons have been engineered by reverse genetics to express reporter proteins, such as EGFP or luciferase, from an additional transcription unit or in frame with viral protein genes (few examples are ^20-23). When these viruses replicate, reporter proteins are produced together with viral proteins themselves. This provides a very quantitative assay to measure viral replication and evaluate the inhibitory activity of candidate molecules. This is particularly true for recombinant viruses expressing luciferase (or other enzymes capable of bioluminescence) since this reporter system exhibits a wide dynamic range with a high sensitivity and virtually no background. Furthermore, there is no excitation light source, thus preventing interference with compound fluorescence²⁴.

Here, we detail a high-throughput protocol to screen chemical libraries for broad-spectrum inhibitors of RNA viruses. Compounds are tested first on human cells infected with a recombinant measles virus (MV) expressing firefly luciferase²⁵ (rMV2/Luc, Figure 1, primary screen). MV belongs to Mononegavirales order, and is often considered as a prototypical member of negative-strand RNA viruses. As such, MV genome is used as a template by the viral polymerase to synthesize mRNA molecules encoding for viral proteins. In the recombinant MV strain called rMV2/Luc, luciferase expression is expressed from an additional transcription unit inserted between P and M genes (Figure 2A). In parallel, compounds are tested for their toxicity on human cells using a commercial luciferase-based reagent that evaluates, by ATP quantification, the number of metabolically active cells in culture (Figure 1, primary screen). Entire chemical libraries can be easily screened with these two assays in order to select compounds that are not toxic and efficiently block MV replication. Then, hits are retested for dose-response inhibition of MV replication, the lack of toxicity as well as for their capacity to impair chikungunya virus (CHIKV) replication (Figure 1, secondary screen). CHIKV is a member of Togaviridae family and its genome is a positive single-strand RNA molecule. As such, it is completely unrelated to measles and compounds inhibiting both MV and CHIKV stand a great chance to inhibit a large panel of RNA viruses. CHIKV nonstructural proteins are directly translated from the viral genome, whereas structural proteins are encoded by transcription and translation of a subgenomic mRNA molecule. Our in vitro replication assay for CHIKV is based on a recombinant strain called CHIKV/Ren, which expresses Renilla luciferase enzyme as a cleaved part of the nonstructural polyprotein through an insertion of the reporter gene between nsP3 and nsP4 sequences²⁶ (Figure 2B). The measure of Renilla luciferase activity allows the monitoring of viral replication at the early stage of CHIKV life cycle.

This high-throughput protocol was used to quickly identify compounds with a suitable profile for broad-spectrum antivirals in a commercial library of 10,000 molecules enriched for chemical diversity. Compounds were essentially following Lipinski's rule of five, with molecular weights ranging from 250 to 600 daltons, and log D values below 5. Most of these molecules were new chemical entities not available in other commercial libraries.

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. Dilute compounds again 1:10 by pipetting 4 μl from dilution plates into 36 μl of DMSO and mixing. Pipette 1 μl into dry wells of white, flat bottom, bar-coded tissue culture 96-wells plates. This second set of daughter plates (D2) will be used to evaluate the inhibition of MV replication by compounds at a final concentration of 2 μM. Store daughter plates at -20 °C until use.

2. Preparation of Cell Cultures to Determine Compound Toxicity

1. Grow HEK-293T cells at 37 °C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) with stabilized L-glutamine and supplemented with 10% fetal calf serum (FCS), streptomycin (100 μg/ml) and penicillin (100 IU/ml). Cells are passaged by trypsinization every 4-5 days, and diluted 1:10 into a fresh flask. Cells should be in log phase for experiments.
2. On the day of the experiment, recover cells by trypsinization and perform cell counting. Pellet cells by centrifugation, and resuspend them at 3 x 10⁵ cells/ml in culture medium. Consider that 12.5 ml of cell suspension are required for each screening plate.
3. Transfer cells to a trough, and dispense 100 μl of cell suspension in D1 plates containing spiked chemical compounds. Cell suspension within the trough is regularly agitated to avoid sedimentation.
4. Spike control wells A1, C1, E1, G1, A12, C12, E12 and G12 with 1 μl of DMSO. Corresponding wells will be used as reference for living cells (no toxicity). Spike control wells B1, D1, F1, H1, B12, D12, F12 and H12 with 1 μl of DMSO and 2.5 μl of a 0.5% IGEPAL solution to kill cells. Corresponding wells will be used as a reference for dead cells (high toxicity).
5. Incubate cells for 24 hr at 37 °C and 5% CO₂.

3. Preparation of Cell Cultures Infected by rMV2/Luc

1. Grow and recover cells as described in 2.1 and 2.2. Again, resuspend cells at 3 x 10⁵ cells/ml in culture medium. Consider that 12.5 ml of cell suspension are required for each screening plate.
2. Optional: supplement culture medium with uridine at 20 μg/ml.
   Note: When screening chemical libraries for viral replication inhibitors, it seems that compounds targeting early steps of pyrimidine biosynthesis pathway are frequently isolated^13,16,27,28. The addition of uridine to culture medium is used to filter out such antiviral molecules. Indeed, this efficiently restores viral replication when upstream enzymes of pyrimidine biosynthesis pathway are blocked.
3. Save 1:10 of cell suspension for control wells with non-infected cells, and proceed to infection with remaining volume.
4. Thaw the appropriate volume of rMV2/Luc stock solution. MV stock solutions are usually at 10⁶-10⁸ infectious particles per ml. Culture must be infected with 0.1 infectious particles of rMV2/Luc per target cells, which corresponds to 0.1 multiplicity of infection (MOI). Note: rMV2/Luc is derived from MV vaccine (Schwarz strain) and is manipulated in a BSL2 environment.
5. Add the virus to cell suspension and mix gently. Transfer infected cells to a trough, and dispense 100 μl of infected cell suspension in columns 2 to 11 of D2 plates containing spiked chemical compounds. Cell suspension within the trough is regularly mixed gently.
6. In columns 1 and 12, dispense 100 μl of non-infected cells a well on two. Infected cells are dispensed in the others wells.
7. Incubate cells for 24 hr at 37 °C and 5% CO₂.

4. Luciferase Activity Measures and Data Analysis

1. Remove D1 plates from the incubator, and determine cellular viability by adding 50 μl of luciferase-based viability reagent directly to culture wells. Mix and incubate for 10 min at room temperature. Read plates with a luminometer. Integration time is set at 100 msec per well.
2. Remove D2 plates from the incubator, and determine luciferase activity by adding 50 μl of luciferase substrate directly to culture wells. Incubate for 6 min at room temperature, and read plates with a luminometer as described above.
3. Calculate a Z'-factor for each D1 plate of the toxicity assay to warrant the quality of the screen²⁹. Z' = 1-3*(σ+ + σ-)/(µ+ - µ-), where µ+ and σ+ correspond to means of luminescence and standard deviations for HEK-293T cells with DMSO alone (no toxicity), and µ- and σ- are means of luminescence and standard deviations for culture wells treated with IGEPAL to kill cells. Z' is expected to be above 0.5, or the corresponding plate is discarded.
4. Set toxicity threshold at µ+/2. Discard compounds with luminescence values below this cut-off (Figure 3A).
5. Calculate a Z'-factor for each D2 plate of the antiviral assay. Here, µ+ and σ+ correspond to means of luminescence and standard deviations for infected cells cultured with DMSO alone, and µ- and σ- are means of luminescence and standard deviations for non-infected cells. Again, plates with Z' values below 0.5 are discarded.
6. Calculate the inhibition of viral replication as follow: percentage of inhibition = (µ+ - luciferase activity for compound X)/(µ+ - µ-)*100. Set inhibition threshold at 75%, and select compounds that both reduce luminescence values below this cut-off (Figure 3B) and are not toxic according to criteria described in 4.4.

5. Secondary Screen for Toxicity and Broad-spectrum Antiviral Activity

1. Make 1:2 serial dilutions for each hit compound in DMSO, starting from 500 µM to 4 µM (8 dilutions). Fill white, bar-coded tissue culture plates with 50 µl of culture medium. Add 1 µl of each compound dilution in culture wells. Repeat twice to have 3 culture plates with duplicate serial dilutions for each hit compound.
2. Prepare 37.5 ml of a HEK-293T cell suspension at 6 x 10⁵ cells/ml in culture medium as described above (in 2.1 and 2.2). Dispense 2x 12.5 ml in falcon tubes and infect cells with either rMV2/Luc at MOI = 0.1 or recombinant CHIKV expressing Renilla luciferase (CHIKV/Ren) at MOI = 0.2. Mix by inverting.
   Note: CHIKV/Ren is derived from a wild-type strain of CHIKV and therefore should be manipulated in a BSL3 environment. Plates can be moved from BSL3 to BSL1 once Renilla substrate has been added to culture wells (see below).
3. Dispense 50 µl of uninfected, rMV2/Luc-infected, or CHIKV/Ren-infected cells in culture plates containing serial dilutions of hit compounds. Incubate 24 hr at 37 °C.
4. Add 50 µl of firefly luciferase substrate to determine firefly luciferase activity in rMV2/Luc-infected wells. Add 50 µl of Renilla luciferase substrate to determine Renilla luciferase activity in CHIKV/Ren-infected wells. Finally, add 50 µl of luciferase-based viability assay reagent to uninfected cells to determine cellular viability.
5. Plot data (Figure 4) and determine hit compound concentrations that inhibit MV and CHIKV replication by 50%. Disregard compound showing some toxicity in this assay.

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 ...

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...

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.