Literature Review: DRAQ5™ in anti-viral therapeutics discovery
DRAQ5™ is widely benchmarked as a tool in both high throughput screening assays for viral infectivity and neutralisation by in-cell western assay™ (ICW), a highly robust semi-quantitative method with a low barrier to entry, and in high content screening (using automated microscopy) as part of the discovery workflow for new or re-purposed anti-viral therapies.
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In-Cell Western Assay™
In principle, the ICW assay utilises cells, which are fixed, permeabilised and stained with antibody (directly fluorescently-tagged or indirectly via a fluorescent secondary antibody) and the signal from the entire well used to monitor expression of a protein or presence, in this case, of a marker of viral infection under different experimental conditions. DRAQ5™, as a stoichiometric reporter of DNA content, is used to normalise the reporter fluorescent signal for variances in cell number between wells correlated to a standard curve of DRAQ5™ fluorescence versus known cell number. Two key references provide full methodological details in virological studies consistent with previous ICW work:
Iskarpatyoti et al. (2012) compared ICW as a high throughput and statistically robust alternative to fluorescence microscopy. They studied antibody blocking of cell infection by reovirus with isolated anti-viral human IgA and by rotavirus with sero-specific monoclonal antibodies and detected with primary polyclonal antisera and fluorescently-tagged (IRDye™ 800 CW) secondary antibodies. Cell nuclei were labelled with DRAQ5™ and Sapphire 700™. Note: This combination of dyes to report cell number was explained to give a wider linear range but others have demonstrated a full linear range with DRAQ5 alone, at 5 µM (Hannoush RN, 2008 and others), with the obvious streamlining of the protocol. Results were consistent with the expected titration-dependent inhibition of viral cell infection and demonstrate that this method is agnostic of target (adherent) cell type or the corresponding infectious virus.
Meanwhile, Cox et al. (2016) developed a Dengue virus (DENV) neutralisation assay with ICW. Dilutions of volunteer donors’ plasma were used to establish titres that would direct the investigators to donors with promising anti-viral activity. These were then progressed with a view to better understanding of immune response and for the generation of human B cell hybridomas that may assist in identification of antigenic determinants and therefore vaccine development. Donors’ plasma was serially diluted and incubated with selected DENV strains. After a period to permit cell infection, cells were fixed permeabilised and stained with a pre-defined biotinylated monoclonal antibody against DENV to aid the identification of candidates for recovery of B cells from isolated PBMCs. To further streamline the assay streptavidin-coupled IRDye™ 800 CW was pre-mixed with DRAQ5™ for a single incubation for fluorescent labelling to measure the viral protein present and counterstaining of the nuclei for cell enumeration, respectively. This work also provides an approach to the screening of convalescent plasma when that has been shown to have benefit in the particular infection.
In both examples, plates were analysed on LICOR’s Odyssey® imaging system. However, such assays have been performed on the TTP Labtech’s Mirrorball.
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High Content Cell-Based Assays
Despite the obvious simplicity and high throughput nature of ICW the higher biological content available by measuring system perturbation cell-by-cell across a population in a well utilising automated fluorescence microscopy is already broadly adopted and well validated across drug discovery (e.g. oncology, parasitology). Importantly, this does give potential for more complex and also unified readouts for: viral infection and neutralisation; drug efficacy and target ID assays on target cells; cellular compartment-level interrogation; and the hallmarks of toxicity and apoptosis; all by high content imaging and which can be agnostic of the available instrumentation.
Commensurate with this, DRAQ5™ has been employed to provide nuclear counterstaining. A number of important features enable achievement of these complex, unified readouts: demarcation of the nucleus - spectrally-compatible with common, lower-cost fluorophores coupled to antibodies or a GFP-tagged protein; counting of cells (nuclei) – as a measure of cell death or proliferation; nuclear morphology change - as an early indicator of toxicity; image analysis of enlarged or proximal nuclear masses – pointing to syncytia formation; segmentation of both nuclear and cytoplasmic compartments by a two-step process of nucleus intensity mask and cytoplasmic “watershed” algorithm achieved from a single reagent and fluorescence channel;
Viral host cell gene studies:
Two reports used siRNA knockdown assays to identify host genes involved in infection of HIV and DENV, respectively. DRAQ5™ was chosen as counterstain in both cases. In Genovesio et al. (2011), GFP-tagged LTR signalled early HIV infection and its intracellular distribution and intensity were correlated to infection against knockdowns of a human whole genome siRNA library. Morphometric features from the DRAQ5™ counterstaining indicated toxicity, syncytia formation and, most simply, a change in cell number. Kwon et al. (2014) used a well-classified monoclonal antibody and fluorescently-tagged secondary antibody to assess increased / reduced intracellular virus following knockdown by a human kinome siRNA library.
Drug re-purposing for anti-viral response:
Schafer et al. (2018) used high content imaging as part of a strategy to re-purpose legacy pharmaceuticals identifying first generation antihistamines with anti-viral properties against the filovirus, Ebola (EBOV). Target host cells were exposed to EBOV and treated with a compound library. Cells were fixed, probed with EBOV glycoprotein- specific primary antibody, fluorescently-tagged secondary antibody and finally DRAQ5™ staining to enable segmentation of the nucleus and cytoplasm to score infection level for a given compound's anti-viral activity.
Drug combination for better anti-viral efficacy:
Ma et al. (2018) investigated the use of a broad-spectrum anti-viral small molecule drug, favipiravir, to enhance the efficacy of one of the few host-targeted molecules, IHVR 19029 an inhibitor of ER α-glucosidases I & II, against hemorrhagic viruses including EBOV for which a cell-based assay was used, detecting cell infection levels by immunofluorescence using a primary monoclonal antibody against EBOV, a secondary antibody and DRAQ5™ to identify nuclei.
Small molecule discovery:
In the search for novel chemical entities against filoviruses Warren et al. (2016) identified a small molecule GS-5734, a prodrug of an adenosine analogue, with high activity and low toxicity in pathogenic RNA viruses including filoviruses (EBOV), arenaviruses and coronaviruses (MERS). DRAQ5™ was deployed in high content imaging assays for relevant host cell types (U2OS, HeLa, macrophages) and a variety of test viruses with specific murine monoclonal antibodies and a common fluorescently-tagged secondary antibody.
Natural compound discovery:
In the work of Estoppey et al. (2017) DENV-infected A549 cells were exposed to canivafungin, a natural compound, to establish IC50 & CC50. Additionally, HUH7 (Cas9 stably-transfected) cells were exposed to CRISPR/Cas9 genetic knockout and then DENV infected to localise the target or target pathway. In both screens, cells were fixed and probed with fluorescently-tagged primary antibody against flavivirus envelope protein. Thereafter, cells were counterstained with DRAQ5™.
Patient diagnosis & trial monitoring:
Koishi et al. (2018) proposed a high content screening approach to rapid diagnosis of Zika virus (ZIKV) infection. To achieve this, Huh7.5 cells were exposed to virus and inactivated patient sera to thereby screen for presence of anti-viral neutralising antibodies in sera. After incubation, cells were fixed and probed with monoclonal against ZIKV envelope protein and indirectly detected by fluorescently-tagged secondary antibody and finally counterstained with DRAQ5™. This method could also be applied to the monitoring of subjects in vaccine trials.
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References:
Cox, Kara S., et al. "Rapid isolation of dengue-neutralizing antibodies from single cell-sorted human antigen-specific memory B-cell cultures." mAbs. 8.1. (2016): 129-140 .
Estoppey, David, et al. "The natural product cavinafungin selectively interferes with Zika and dengue virus replication by inhibition of the host signal peptidase." Cell Reports 19.3 (2017): 451-460.
Genovesio, Auguste, et al. "Automated genome-wide visual profiling of cellular proteins involved in HIV infection." Journal of biomolecular screening 16.9 (2011): 945-958.
Hannoush, Rami N. "Kinetics of Wnt-driven β-catenin stabilization revealed by quantitative and temporal imaging." PloS one 3.10 (2008): e3498.
Iskarpatyoti, Jason A., et al. "A rapid, automated approach for quantitation of rotavirus and reovirus infectivity." Journal of virological methods 184.1-2 (2012): 1-7.
Koishi, Andrea Cristine, et al. "Development and evaluation of a novel high-throughput image-based fluorescent neutralization test for detection of Zika virus infection." PLoS neglected tropical diseases 12.3 (2018): e0006342.
Kwon, Yong-Jun, et al. "Kinome siRNA screen identifies novel cell-type specific dengue host target genes." Antiviral Research 110 (2014): 20-30.
Ma, Julia, et al. "Enhancing the antiviral potency of ER α-glucosidase inhibitor IHVR-19029 against hemorrhagic fever viruses in vitro and in vivo." Antiviral research 150 (2018): 112-122.
Schafer, Adam, et al. "Repurposing potential of 1st generation h1-specific antihistamines as anti-filovirus therapeutics." Antiviral research 157 (2018): 47-56.
Warren, Travis K., et al. "Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys." Nature 531.7594 (2016): 381-385.
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