High-throughput screening applications on microplate readers

384-well microplate

This article is sponsored by BMG Labtech.

High-throughput screening (HTS) is predominantly used in drug discovery and relevant to the pharmaceutical industry as well as academic core facilities. With HTS, researchers can quickly test large libraries of chemical, biochemical, or pharmacological samples for drug candidates.

To maximise efficiency, screening facilities must constantly strive to increase throughput and minimise sample expenditure, while controlling costs. This leads to the key factors in HTS: highest speed and flexibility without compromising on sensitivity. Microplate readers are an important and indispensable tool for carrying out HTS and are used to increase the overall throughput of a campaign. Single microplates can accommodate up to 3456 microwells, allowing to run the same number of samples per measurement run. Another advantage of high-density microplates is the low filling volume per well which helps reducing the consumption of valuable analytes and expensive assay components. Since HTS campaigns can comprise large compound libraries with millions of test compounds, sophisticated HTS setups including microplate readers and other automation devices save time and money.

But not only devices need to fulfill specific requirements, assays need to be HTS-compatible as well. HTS applications need to be miniaturized to match smaller microplate formats and optimise throughput, but also need to be compatible with lab automation. This requires easy preparation steps that can be covered by liquid handling and a stable performance with only low variability. Not all applications are suited for HTS approaches due to insufficient signal output or high complexity. Over time different microplate reader-based methods have been established that now form the hallmarks of HTS campaigns. These applications often detect interaction events between a relevant target and its binding partner(s). HTS campaigns are used to screen for modulators or competitors of such interactions.

Figure 1: Tracer characterisation. Association was monitored by TR-FRET after combining terbium-labelled kinase and a fluorescent kinase tracer. Dissociation was recorded after addition of excess staurosporine to the tracer sample.

One commonly used approach is Time-Resolved Förster´s Resonance Energy Transfer (TR-FRET). TR-FRET describes the energy transfer between two fluorophores and is regularly employed to screen for interactions between potential drugs and their targets. While TR-FRET offers a simple add-mix-measure principle, it also requires the labeling of the two interaction partners. To streamline this process, labelled targets and tracer molecules are employed. By performing kinetic binding measurements on a microplate reader (Figure 1), the association and dissociation behaviour of the fluorescent tracer towards the target can be determined. Once binding parameters for the tracer molecule are established, this can be used in HTS approaches to screen for compounds competing with the tracer-target interaction.

TR-FRET is not typically suited for studies in live cells and is mainly used in a biochemical setting, using purified proteins in solution or cell lysates. Ratiometric luminescence-based approaches like BRET are the first choice for HTS analyses with live cells,

While ratiometric readouts are often used due to the easy preparation steps and stable assay performance, applications where only one fluorophore or luminophore is used for detection are regularly used in HTS today. Such applications utilise different approaches to investigate binding events such as protein complementation using luminescence or fluorescence polarisation (FP).

Figure 2: Graphical representation of the FP raw data from compound titration against LC3A in 5 µl reactions in a 1536-well plate. Decreasing curves mark tracer displacement and therefore competitor binding. Shown is a titration of 96 compounds in replicates.

FP applications utilise polarised excitation light and measure the polarization of emission light of a fluorescent tracer molecule for this purpose. If the smaller tracer molecule is bound to the larger target of interest this complex has a slow tumbling rate and thus emits light in the same plane as the excitation source resulting in high polarisation values (mP). HTS approaches use FP to screen for competitors of the tracer-target interaction (Figure 2). By adding increasing concentrations of competitors, the fluorescent tracer is displaced. This results in a drop in mP values since the free tracer molecule has a fast tumbling rate and accordingly emits unpolarised light on different polarisation planes.

To meet the requirements of HTS applications BMG Labtech developed the PHERAstar FSX multi-mode microplate reader which is by now the gold standard reader in this field. The reader was specifically conceived for the fastest read times, the best sensitivity and unmatched flexibility in all plate formats up to 3456 wells. It offers a unique combination of features to support all major HTS applications. The PHERAstar FSX easily performs applications like protein-protein interactions, affinity binding assays, compound and inhibitor screening and can be easily automated in robotic systems, making it the ideal measurement platform for any HTS campaign.

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