ReviewNew-generation screening assays for the detection of anti-influenza compounds targeting viral and host functions
Introduction
The use of high-throughput screening (HTS) technology for antiviral discovery is a fairly recent endeavor, first undertaken exclusively by the pharmaceutical industry and now also performed by academic scientists. The development of HTS has been driven by increasing advances in automation and the ability to handle large datasets. It has also expanded the types of target that can be explored and consequently assay development, particularly of cell-based assays, is a major part of all antiviral HTS campaigns.
As a small RNA virus, influenza virus encodes a limited number of proteins and thus there are only a few viral functions that are considered to be tractable drug targets by traditional standards. This essentially means that the target must have a function that is amenable to inhibition by a small molecule. The current two classes of approved antivirals for influenza target either the ion channel function of the M2 protein or the neuraminidase function of the NA protein. The neuraminidase inhibitors (NAI) were developed through the rational design of small molecules that mimic sialic acid and bind with high affinity to the active site of NA (Gubareva et al., 2000). The adamantanes are an interesting example of an antiviral whose approval preceded knowledge of the target (M2) or the function of the target as an ion channel. Moreover, the precise mechanism of action is still under debate following publication of structures showing different placement of the drug relative to M2 (Cady and Hong, 2008, Cady et al., 2010, Pielak and Chou, 2010, Stouffer et al., 2008). Other well-characterized viral functions that should be druggable are the RNA-dependent RNA polymerase activity of PB1 and the endonuclease function of PA. Apart from the fact that the description of PA endonuclease activity was only made in 2009 (Dias et al., 2009, Yuan et al., 2009), the major reason that these targets have not been explored fully is the inability to produce purified, full-length and active polymerase proteins, which severely limits the development of biochemical screening assays.
The examples above refer to viral functions that are considered to be validated targets, as it is known a priori that they are essential for influenza virus growth, and biochemical assays can (or could) be developed to screen for specific inhibitors of that function. Alternatively, one can cast a wider net by not requiring knowledge of the target or function upfront and instead using a phenotypic readout such as virus replication. This approach requires a cell-based assay and it is in this area that we have seen most development in the influenza virus HTS field. The advantages are: (i) that it potentially allows one to capture all stages of the virus life-cycle in one assay, (ii) it detects inhibitors of cellular functions that are required for virus replication, and (iii) it may reveal unknown functions of viral proteins that are susceptible to small molecule inhibition. This review will focus on the new tools that have been developed for influenza antiviral drug discovery, with an emphasis on the use of fluorescent or luminescent reporters and the development of novel cell-based assays.
Section snippets
Suitable HTS assays for influenza antiviral discovery
The type of assay chosen for a screen depends on the question being asked and what tools are available. If the purpose is to identify inhibitors of as many different steps of the influenza virus life-cycle as possible, then an assay involving virus infection of cells must be used, preferably under conditions of multi-cycle replication (see Section 3.1). The readout for this type of assay can vary from antibody-based detection of viral proteins, to expression of reporter genes encoded by the
Single versus multi-cycle viral replication assays
When designing an assay to monitor influenza virus replication it is important to understand the concept of single cycle vs. multi-cycle replication as this affects the stages of the virus life-cycle that can be captured by the assay. In a single cycle assay, 100% of cells are infected in the first round and thus this type of assay is performed with a high multiplicity of infection (MOI). If the assay readout is viral gene expression, this assay will capture all steps from virus attachment
Biochemical assays
Cell-free biochemical assays for drug discovery come with the benefits of shorter duration, the absence of toxicity issues, simpler experimental conditions (there is no need for sterile technique, for example) and amenability to HTS and automation. For these reasons cell-free systems have often been the starting point in drug-screening projects, however not all biological activities can be studied in this way.
Summary
As described above, there have been significant advances in the development of tools for influenza virus HTS assays in recent years. In particular, the generation of recombinant, reporter-expressing viruses that are replication competent allows for the design of cell-based assays that capture all stages of the virus life-cycle. With these viruses there is greater flexibility in the choice of cells for the assay, so together this provides increased potential for identifying inhibitors of both
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These authors contributed equally to this work.