Developing robust and reliable high-throughput screening assays
High-throughput screening (HTS) plays a pivotal role in drug discovery. It’s used to hunt for ‘hits’ – those sought-after molecules that modulate the activity of targeted biological pathways. Hits can progress through the pipeline to lead compounds and potentially, new medicines. Screening for hits is an important first step that relies on making the right decision at every stage of the process.
Hit identification campaigns begin with the design of suitable screening assays. These must be tailored to the target, and a number of key factors should be considered to ensure these are biologically relevant, reliable and robust.
For example, will you use biochemical or cell-based assays? Biochemical assays screen compounds against an isolated target in an artificial environment, whereas cellular assays have the advantage of testing compounds in a pristine physiological setting. While cell-based assays tend to avoid some of the common artifacts encountered in biochemical assays, they can falsely identify hits that do not act on the target of interest, and may miss hits that do not penetrate the cell membrane.
For enzyme assays, it’s important to consider whether full length proteins or peptide fragments will be used. Similarly, while recombinant cell lines are commonly used in cell-based assays, there may be instances where wild type cell lines are more beneficial.
Next is the choice of assay detection method. The most common detection techniques for biochemical assays are optical (absorbance, fluorescence or luminescence), while radiometric or electrophysiological techniques can also be used. Label-free detection assays are becoming more commonplace, thanks to advances in technologies such as mass spectrometry and calorimetry.
Often, multiple detection techniques are amenable to a given target and matching a detection technique to a particular target comes down to a tradeoff between ease of development, predictability, relevance and cost. Practical factors such as miniaturization can also be important considerations.
Building a focused screening compound library
To ensure screening campaigns run as efficiently as possible, it’s important to consider the compounds that will be screened. A thorough understanding of the competitor landscape and knowledge of the target classes’ history of druggability can help to guide this selection process and avoid wasted efforts.
Computational chemistry, based on the knowledge of binding site structure obtained through crystallography, also plays an important role in compound selection. Ligand and structure-based virtual screening can help to direct the choice of a screening deck, and build and test binding hypotheses. Identified hits can also undergo virtual ADME toxicokinetics studies to further build a picture of the structures that can be taken forward to the screening stage.
Of course, it’s important to ensure that your screening collection is diverse and covers a large area of chemical space. This should include structural, chemical and stereochemical diversity, to ensure molecules have a sufficiently broad range of shapes, sizes and pharmacophores (binding properties) that will allow them to interact with the target’s binding site. Care should also be taken around promiscuous moieties – false positives that result from interference with the assay detection technology or non-specific interaction with the target protein.
Adopting a multidisciplinary, considered screening strategy
When embarking on an HTS campaign, it’s often best to start with a small pilot screen. This can be used to confirm the developed assay behaves as expected, and help to determine a suitable substrate concentration.
Then there’s the scale on which to run your assay. Ultra-HTS enables rapid testing of over a million samples in less than two weeks. Alternatively, a more iterative approach can be adopted, where two or three smaller screens are run with intermediate data analysis. The latter approach can help to focus HTS where screening is initially open to a structurally broad screening collection.
Of course, the goal of the hit-to-lead phase is to obtain qualified hits – that is, hits that have demonstrated activity against a target and can be taken forward as strong lead candidates. An initial hit series may contain artifacts and promiscuous compounds, and secondary assays to confirm activity form an essential part of any hit identification campaign.
Here, at least one additional related biological target should be tested. It’s also important to perform orthogonal testing where an alternative detection approach (e.g. electrophysiological) is complemented with another (e.g. fluorescence). This helps to boost confidence that the observed activity of the compound is due to binding with the target, and not due to interference with the assay detection technology.
Structure-activity relationship (SAR) studies can be undertaken to further develop the binding hypothesis and knowledge of active regions in chemical space. This SAR expansion can be used to find derivatives of active scaffolds from low-activity data.
Hit identification draws on the expertise of biologists, chemists, engineers and informatics specialists. To find out more about how Aptuit’s experts take an integrated approach to drug discovery, watch our webinar.