Development of a homogeneous screening assay for automated detection of antiviral agents active against severe acute respiratory syndrome-associated coronavirus

Ivens, Tania; Eynde, Christel Van den; Acker, Koen Van; Nijs, Erik; Dams, Géry; Bettens, Eva; Öhagen, Åsa; Pauwels, Rudi; Hertogs, Kurt

doi:10.1016/j.jviromet.2005.05.010

Cited by 39 publications

(56 citation statements)

References 25 publications

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“…De Clercq's group has pioneered in developing a facile and automated assay based on the bioreduction of the tetrazolium salts 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for the detection of anti-HIV compounds out of a large compound library using an inexpensive cell-based assay [21]. Several groups have also adopted this strategy to identify initial hit compounds based on respective antiviral targets as a starting point for drug discovery against SARSCoV [16,[22][23][24].…”

Section: High Throughput Screening (Hts) Methodsmentioning

confidence: 99%

“…Copper ion weakly inhibited the activity of PLP2 at 10 µM to 70%, and two zinc complexes (22,38), previously reported active against SARS-CoV M pro , also inhibited SARS-CoV PLP2 activity with the IC 50 value of 3.3 and 3.7 µM respectively [39]. The inhibition was specific because other divalent metals such as Mg, Mn, Ca, Ni and Co had no effects on the activity of SARS-CoV PLP2 at 10 µM.…”

Section: Helicase Inhibitor 322 Papain-like Protease 2 Inhibitorsmentioning

confidence: 97%

“…A panel of metal ions including Zn 2+ and its conjugates were also evaluated for their anti-SARS-CoV M pro activities. Inhibition was more pronounced using a zinc-conjugated compound (1-hydroxypyridine-2-thione zinc (22); K i = 0.17µM) than using the ion alone (K i = 1.1 µM). Two other compounds, tannic acid (23) and 3-isotheaflavin-3-gallate (TF2B) (24a), belonging to a group of polyphenols isolated from tea were also found to inhibit the SARS M pro activity.…”

Section: (B) Non Peptide Compoundsmentioning

confidence: 98%

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Antiviral Drug Discovery Against SARS-CoV

Wu¹,

Lin²,

Hsu³

et al. 2006

CMC

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Severe Acute Respiratory Syndrome (SARS) is a life-threatening infectious disease caused by SARS-CoV. In the 2003 outbreak, it infected more than 8,000 people worldwide and claimed the lives of more than 900 victims. The high mortality rate resulted, at least in part, from the absence of definitive treatment protocols or therapeutic agents. Although the virus spreading has been contained, due preparedness and planning, including the successful development of antiviral drugs against SARS-CoV, is necessary for possible reappearance of SARS. In this review, we have discussed currently available strategies for antiviral drug discovery and how these technologies have been utilized to identify potential antiviral agents for the inhibition of SARS-CoV replication. Moreover, progress in the drug development based on different molecular targets is also summarized, including 1) Compounds that block the S protein-ACE2-mediated viral entry; 2) Compounds targeting SARS-CoV M(pro); 3) Compounds targeting papain-like protease 2 (PLP2); 4) Compounds targeting SARS-CoV RdRp; 5) Compounds targeting SARS-CoV helicase; 6) Active compounds with unspecified targets; and 7) Research on siRNA. This review aims to provide a comprehensive account of drug discovery on SARS. The experiences with the SARS outbreak and drug discovery would certainly be an important lesson for the drug development for any new viral outbreaks that may emerge in the future.

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Section: High Throughput Screening (Hts) Methodsmentioning

confidence: 99%

Section: Helicase Inhibitor 322 Papain-like Protease 2 Inhibitorsmentioning

confidence: 97%

Section: (B) Non Peptide Compoundsmentioning

confidence: 98%

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Antiviral Drug Discovery Against SARS-CoV

Wu¹,

Lin²,

Hsu³

et al. 2006

CMC

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“…In addition the antiviral activity of a known SARS inhibitor, that is, Anti-ACE2 IgG was evaluated. This antibody inhibits SARS virus replication by binding ACE2 and preventing the virus to enter the cell (Li et al, 2003;Ivens et al, 2005). The experiment was set-up as a standard antiviral experiment in which the antibody was diluted in medium at different concentrations in duplicate after which virus and cells were added.…”

Section: Bis-aaf-r110 Cleavage (Fluoresence Rfu)mentioning

confidence: 99%

A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE

Jochmans

Leyssen

Neyts

2012

Journal of Virological Methods

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For antiviral screenings purposes, infection of cell cultures with the virus under study, should ideally result in the induction, within just a few days, of (nearly) complete CPE and allow the calculation of acceptable Z' factors (>0.5). The human Corona virus NL63 (HCoV-NL63) causes only limited CPE on different cell lines (Schildgen et al., 2006). Following infection of Vero118 cells, virus-induced CPE was too low to allow readout based on classical colorimetric methods (such as the MTS assay), even following prolonged incubation times (>7 days). To develop an antiviral screenings-assay against HCoV-NL63, we explored whether a dead-cell protease substrate could be used instead. The substrate used is a quenched peptide (bis-AAF-R110) that releases a fluorophore upon proteolytic-cleavage by proteases; the latter released from dead cells. Following different rounds of optimization a screening protocol was developed: Vero118 cells in 96-well plate format were infected with HCoV-NL63 (MOI=0.01; 200μL cell culture; 2.10(4)cells/mL, IMDM 5% FBS medium). Cultures were subsequently incubated for 5 days at 35°C after which 20μL of the peptide solution was added. Fluorescence was quantitated 2 hr after incubation at 37°C. A roughly 3-fold increase in fluorescence intensity in the infected cultures was observed as compared to the uninfected cultures with a low well-to-well variability. Z' factors calculated from different experiments were in the range of 0.6-0.8, indicating excellent assay quality. An anti-ACE2 polyclonal antiserum (that prevents coronavirus infection in cell cultures) was used as a positive control and allowed to validate the assay for antiviral screening purposes. In conclusion, in conditions where a viability staining is inadequate to quantitate virus-induced CPE, a novel simple and convenient method that detects cell-death and that is suitable for high-throughput screening purposes can be employed.

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“…SARS-CoV drug discovery requires the development of reliable biological assays. Traditional antiviral assays for coronavirus are based on viral infection of cultured cells, followed by monitoring of compound inhibition of viral replication through observation of cytopathic effects, quantification of viral yields by plaque assay, or measurement of viral RNA by reverse transcription-PCR (Ivens et al, 2005;Wu et al, 2004). The virus can infect many cell types and produces plaques (Hofmann and Pöhlmann, 2004).…”

Section: Introductionmentioning

confidence: 99%

High-throughput assay using a GFP-expressing replicon for SARS-CoV drug discovery

Xiong

Lin

et al. 2008

Antiviral Research

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The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The development of rapid screening assays is essential for antiviral drug discovery. By using a cell line expressing a SARS-CoV subgenomic replicon, we developed a high-throughput assay and used it to screen small molecule compounds for inhibitors of SARS-CoV replication in the absence of live virus. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. Based on this assay system, we screened 7035 small molecule compounds from which we identified 7 compounds with anti-SARS-CoV activity. We demonstrate that the compounds inhibited SARS-CoV replication-dependent GFP expression in the replicon cells and reduced SARS-CoV viral protein accumulation and viral RNA copy number in the replicon cells. In a SARS-CoV plaque reduction assay, these compounds were confirmed to have antiviral activity. The target of one of the hit compounds, C12344, was validated by the generation of resistant replicon cells and the identification of the mutations conferring the resistant phenotype. These compounds should be valuable for developing anti-SARS therapeutic drugs as well as research tools to study the mechanism of SARS-CoV replication.

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Development of a homogeneous screening assay for automated detection of antiviral agents active against severe acute respiratory syndrome-associated coronavirus

Cited by 39 publications

References 25 publications

Antiviral Drug Discovery Against SARS-CoV

Antiviral Drug Discovery Against SARS-CoV

A novel method for high-throughput screening to quantify antiviral activity against viruses that induce limited CPE

High-throughput assay using a GFP-expressing replicon for SARS-CoV drug discovery

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